Antibodies specific to delta 1 chain of T cell receptor

ABSTRACT

Disclosed herein are antibodies specific to a delta1 chain of a γδ T cell receptor and methods of using such for modulating γδ T cell bioactivity. Such anti-Delta1 antibodies may also be used to treat diseases associated with γδ T cell activation, such as solid tumors, or for detecting presence of γδ1 T cells.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119 of United States provisional application Nos. 62/620,813, filed Jan. 23, 2018, 62/736,321, filed Sep. 25, 2018, and 62/779,915, filed Dec. 14, 2018, the contents of each of which are incorporated by reference herein in their entirety.

BACKGROUND OF INVENTION

Immune checkpoint blockade has demonstrated unprecedented success in the past few years as cancer treatment. Often antibodies are used to block immune inhibitory pathways, such as the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1) pathways. While therapies targeting those two pathways have shown success in treating several cancer types, anti-CTLA-4 and anti-PD-1 therapies have a response rate of 10 to 60% of treated patients, depending on cancer type, and have not yet shown the ability to exceed a response rate of 60%, even when used in combination (Kyvistborg et al., Enhancing responses to cancer immunotherapy; Science. 2018 Feb. 2; 359(6375):516-517). Additionally, a large number of cancer types are refractory to these therapies.

γδ T cells are a subgroup of T cells which have distinct T cell receptor (TCR) γ and δ chains on their surface. This sets them apart from CD4+ helper T cells and CD8+ cytotoxic T cells, which express αβ TCRs on their cell surfaces. Recent studies have found that γδ T cells have protumor activity (Zhao et al. J Transl Med (2018) 16:3). For example, in human pancreatic ductal carcinoma, γδ T cells have been found to constitute a substantial fraction of tumor-infiltrating T cells and to inhibit the anti-cancer immune response mediated by alpha beta (αβ) T cells (Daley et al., Cell, 2016, 166: 1485-1499). In the tumor microenvironment (TME), γδ T cells have been shown to express IL-4, IL-10, and TGF-β, leading to suppression of the anti-tumor response (Kuhl et al., Immunol., 2009, 128(4): 580-588). The expression of both IL-10 and TGF-β has been shown to be increased in a variety of cancer types (Lafont et al., Front Immunol., 2014, 5: 622). γδ T17 cells are a major source of IL-17 in the tumor microenvironment, where they function to promote angiogenesis in a number of cancer types (Silva-Santos B. Eur J Immunol. 2010; 40:1873-6; Zhao et al. J Transl Med (2018) 16:3, and references therein). Additionally, γδ T cells have been found to induce senescence of naïve and effector T cells, which become suppressive and increase immunosuppression in the TME (Ye et al., J Immunol., 2013, 190(5): 2403-2414). Finally, studies have shown that γδ T cells increase the presence of myeloid derived suppressor cells (MDSCs) in the TME, promoting a pro-tumor microenvironment (Yan and Huang, Oncoimmunology. 2014; 3:e953423; Qu P, et al., Cancer Lett. 2016; 380:253-6, and references therein).

Given the average response rate and the large number of cancer types that are refractory to current treatment, there remains a need for new cancer therapies. Modulating the activity of gamma delta T cells and/or one or more of its T cell receptors provides a novel cancer therapy approach.

SUMMARY OF INVENTION

The modulation of gamma delta T cell activity and/or one or more of its T cell receptors may be used alone or in combination with existing therapies as a means for cancer treatment. Described herein are novel human antibodies which bind to human gamma delta T cell receptors and their therapeutic use in the treatment of cancer. The present disclosure is based, at least in part, on the development of antibodies that specifically bind to delta1 chains of γδ T cell receptors (TCRs). Such antibodies are capable of inhibiting the immune suppression mediated by the γδ T cells.

Accordingly, one aspect of the present disclosure provides an isolated antibody, which specifically binds a delta1 chain of a T cell receptor. In some embodiments, the antibody cross-reacts with γδ TCRs having different delta1 chain sequence. In some embodiments, the antibody binds to a γ61 TCR having a specific 61 chain as relative to γ61 TCRs containing 61 chain having different sequence.

In some embodiments, any of the isolated anti-Delta1 antibodies described herein may bind a T cell receptor comprising a delta1 chain and a gamma chain. The gamma chain of the T cell receptor can be any gamma chain, including but not limited to e.g., gamma-1, gamma-2, gamma-3, gamma-4, gamma-5, gamma5P, gamma-8, gamma-9, gamma-10, gamma-11, and/or gamma-a. Gamma chains are encoded by TRGV1, TRGV2, TRGV3, TRGV4, TRGV5, TRGV5P, TRGV8, TRGV9, TRGV10, TRGV11, and TRGVA genes. In some embodiments, the antibody is specific to a specific delta1 chain (e.g., having specified sequence) and a specific gamma chain (e.g., having specified sequence). In some embodiments, the antibody is specific to a specific delta1 chain and has cross-reactivity to more than one gamma chain (e.g., binds to 2, 3, 4, 5, 6, 7, 8, or 9 etc. gamma chains or binds universally to all gamma chains). In some embodiments, the antibody has cross-reactivity to more than one delta1 chain sequence and cross-reactivity to more than one gamma chain. In some embodiments, the antibody is specific to γ9δ1 TCR (containing a delta1 chain and a gamma9 chain). In some embodiments, the anti-Delta1 antibody cross-reacts with γδ1 TCRs containing different gamma chains (e.g., gamma-1, gamma-2, gamma-3, gamma-4, gamma-5, gamma5P, gamma-8, gamma-9, gamma-10, gamma-11, and/or gamma-a). In some embodiments, the anti-Delta1 antibody cross-reacts with γδ1 TCRs containing different gamma chains (e.g., as encoded by TRGV1, TRGV2, TRGV3, TRGV4, TRGV5, TRGV5P, TRGV8, TRGV9, TRGV10, TRGV11, and TRGVA).

In some embodiments, the anti-Delta1 antibody preferentially binds a γδ1 TCR as relative to a γδ2 TCR or a or γδ3 TCR. In one particular embodiment, the anti-Delta1 antibody does not bind a T cell receptor comprising a delta-2 chain and a gamma chain.

In some embodiments, the anti-Delta1 antibody disclosed herein binds a human delta1 chain. In some embodiments, the anti-Delta1 antibody disclosed herein is selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 antibodies. Each of these antibodies are referred to herein as a “reference antibody”. In some embodiments, the anti-Delta1 antibody disclosed herein binds to the same epitope as any one of the Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17 antibodies and/or competes against any of the just-noted reference antibodies from binding to the epitope.

In some embodiments, the anti-Delta1 antibody comprises a heavy chain variable domain (V_(H)) comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3), which collectively are at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the heavy chain CDRs of a reference antibody; and/or the antibody comprises a light chain variable domain (V_(L)) comprising a light chain CDR1, a light CDR2, and a light chain CDR3, which collectively are at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the light chain CDRs of a reference antibody.

In some embodiments, the anti-Delta1 antibody comprises a heavy chain variable domain (V_(H)) comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3), which collectively are at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the heavy chain CDRs of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 antibodies; and/or the antibody comprises a light chain variable domain (V_(L)) comprising a light chain CDR1, a light CDR2, and a light chain CDR3, which collectively are at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the light chain CDRs of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 antibodies.

In some embodiments, the anti-Delta1 antibody comprises the same heavy chain complementarity determining regions (CDRs) and the same light chain CDRs as a reference antibody. In some embodiments, the anti-Delta1 antibody comprises the same heavy chain variable region and the same light chain variable region as an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 antibodies.

In one specific embodiment, the anti-Delta1 antibody comprises a heavy chain variable domain (V_(H)) comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3) identical to the heavy chain CDRs of Delta1-17. In some embodiments, the anti-Delta1 antibody comprises a light chain variable domain (V_(L)) comprising a light chain CDR1, a light CDR2, and a light chain CDR3, identical to the light chain CDRs of Delta1-17. In some embodiments, the anti-Delta1 antibody comprises a heavy chain variable domain (V_(H)) comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3), which collectively are at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the heavy chain CDRs of Delta1-17; and/or the antibody comprises a light chain variable domain (V_(L)) comprising a light chain CDR1, a light CDR2, and a light chain CDR3, which collectively are at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the light chain CDRs of Delta1-17.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46. In some embodiments, the anti-Delta1 antibody comprises a VL CDR2 having the sequence of SEQ ID NO: 47. In some embodiments, the anti-Delta1 antibody comprises a VL CDR3 having a sequence selected from any of SEQ ID NOs: 48, 52, 56, 59, 63, 67, 70, 74, and 78. In some embodiments, the anti-Delta1 antibody comprises a VL CDR3 having the sequence of SEQ ID NO: 78. In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, and a VL CDR3 having a sequence selected from any of SEQ ID NOs: 48, 52, 56, 59, 63, 67, 70, 74, and 78. In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, and a VL CDR3 having the sequence of SEQ ID NO: 78. In some embodiments, the anti-Delta1 antibody comprises a light chain variable domain (V_(L)) comprising a light chain CDR1, a light CDR2, and a light chain CDR3, which collectively are at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the light chain CDRs of SEQ ID NOs: 46, 47, and 78, respectively.

In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having a sequence selected from any of SEQ ID NOs: 43, 49, 53, 60, and 64. In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 43. In some embodiments, the anti-Delta1 antibody comprises a VH CDR2 having a sequence selected from any of SEQ ID NO: 44, 50, 54, 57, 61, 65, 68, and 72. In some embodiments, the anti-Delta1 antibody comprises a VH CDR2 having the sequence of SEQ ID NO: 57. In some embodiments, the anti-Delta1 antibody comprises a VH CDR3 having a sequence selected from any of SEQ ID NOs: 45, 51, 55, 58, 62, 66, 69, 71, 73, 75, 76, and 77. In some embodiments, the anti-Delta1 antibody comprises a VH CDR3 having the sequence of SEQ ID NO: 77. In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having a sequence selected from any of SEQ ID NO: 43, 49, 53, 60, and 64, a VH CDR2 having a sequence selected from any of SEQ ID NO: 44, 50, 54, 57, 61, 65, 68, and 72, and a VH CDR3 having a sequence selected from any of SEQ ID NOs: 45, 51, 55, 58, 62, 66, 69, 71, 73, 75, 76, and 77. In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 43, a VH CDR2 having the sequence of SEQ ID NO: 57, and a VH CDR3 having the sequence of SEQ ID NO: 77. In some embodiments, the anti-Delta1 antibody comprises a light chain variable domain (V_(L)) comprising a light chain CDR1, a light CDR2, and a light chain CDR3, which collectively are at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the light chain CDRs of SEQ ID NOs: 43, 57, and 77, respectively.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having a sequence selected from any of SEQ ID NOs: 48, 52, 56, 59, 63, 67, 70, 74, and 78, a VH CDR1 having a sequence selected from any of SEQ ID NOs: 43, 49, 53, 60, and 64, a VH CDR2 having a sequence selected from any of SEQ ID NO: 44, 50, 54, 57, 61, 65, 68, and 72, a VH CDR3 having a sequence selected from any of SEQ ID NOs: 45, 51, 55, 58, 62, 66, 69, 71, 73, 75, 76, and 77. In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NO: 78, and a VH CDR1 having the sequence of SEQ ID NO: 43, a VH CDR2 having the sequence of SEQ ID NO: 57, and a VH CDR3 having the sequence of SEQ ID NO: 77. In any of these embodiments, the anti-Delta1 antibody binds to delta1. In some embodiments, the anti-Delta1 antibody comprises a HC CDR1, a HC CDR2, and a HC CDR3, which collectively contains no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid variations relative to the HC CDRs of the reference antibody; and/or wherein the antibody comprises a LC CDR1, a LC CDR2, and a LC CDR3, which collectively contains no more than 8 (e.g., no more than 7, 6, 5, 4, 3, 2, or 1) amino acid variations relative to the light chain CDRs of the reference antibody.

In some embodiments, the anti-Delta1 antibody comprises a HC CDR1, a HC CDR2, and a HC CDR3, which collectively contains no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid variations relative to the HC CDRs of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17; and/or wherein the antibody comprises a LC CDR1, a LC CDR2, and a LC CDR3, which collectively contains no more than 8 (e.g., no more than 7, 6, 5, 4, 3, 2, or 1) amino acid variations relative to the light chain CDRs of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17.

In some embodiments, the anti-Delta1 antibody comprises a HC CDR1, a HC CDR2, and a HC CDR3, which collectively contains no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid variations relative to the HC CDRs of Delta1-17; and/or wherein the antibody comprises a LC CDR1, a LC CDR2, and a LC CDR3, which collectively contains no more than 8 (e.g., no more than 7, 6, 5, 4, 3, 2, or 1) amino acid variations relative to the light chain CDRs of Delta1-17.

In some embodiments, the anti-Delta1 antibody comprises a V_(L) that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(L) of the reference antibody, and/or a V_(H) that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(H) of the reference antibody.

In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(H) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 antibodies, and/or a V_(L) sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the V_(L) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 antibodies.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(L) sequence of Delta1-17, and/or a V_(H) that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(H) of Delta1-17 antibody.

In some embodiments, the anti-Delta1 antibody disclosed herein has the same VL sequence as an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 antibodies. In some embodiments, the anti-Delta1 antibody disclosed herein is an antibody having the same VL sequence as an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 antibodies. In some embodiments, the anti-Delta1 antibody disclosed has the same V_(H) sequence and the same VL sequence as an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 antibodies.

In some embodiments, the anti-Delta1 antibody is Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17. In one specific embodiment, the anti-Delta-1 antibody is Delta1-17.

In some embodiments, the anti-Delta1 antibody comprises a VL region having a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24. In some embodiments, the anti-Delta1 antibody comprises a VH region having a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the anti-Delta1 antibody comprises a VL region a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24 and a VH region having a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 24. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 23. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 24 and a VH region having the sequence of SEQ ID NO: 23.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80% or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18, and 24. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80% or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of or consisting of a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of or consisting of a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80% or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the isolated antibody has a V_(L) sequence comprising a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24 and a V_(H) sequence comprising a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24 and a V_(H) sequence consisting essentially of a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting of a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24 and a V_(H) sequence consisting of a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80% or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 8′7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 24. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80% or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 8′7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 23. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising SEQ ID NO: 24. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of or consisting of SEQ ID NO: 24. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of or consisting of SEQ ID NO: 23.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 24 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 23. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 24 and a V_(H) sequence comprising SEQ ID NO: 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 24 and a V_(H) sequence consisting essentially of SEQ ID NO: 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 24 and a V_(H) sequence consisting of SEQ ID NO: 23. In some examples, the anti-Delta1 antibody may comprise the same heavy chain complementarity determining regions (CDRs) and the same light chain CDRs as the reference antibody. In one specific example, the anti-Delta1 antibody comprises the same heavy chain variable region and the same light chain variable region as the reference antibody. Any of the anti-Delta1 antibodies described herein may be a full-length antibody (e.g., an IgG molecule) or an antigen-binding fragment thereof. In some examples, the antibody is a Fab, a F(ab′)2, or a single-chain antibody. In any instances, the antibody can be a human antibody or a humanized antibody. In some embodiments, the antibody is an antibody drug conjugate. In some embodiments, the antibody is an antibody mimetic.

In another aspect, the present disclosure provides an isolated nucleic acid or set of nucleic acids which encode or collectively encode any of the anti-Delta1 antibodies disclosed herein. In some instances, the heavy chain and light chain of the antibody are encoded by two separate nucleic acid molecules (a set of nucleic acids). In other instances, the heavy chain and light chain of the antibody are encoded by one nucleic acid molecule, which may be in multicistronic format, or under the control of distinct promoters. In some embodiments, the nucleic acid or set of nucleic acids are located on one or two vectors, for example, the one or two vectors may be one or two expression vectors. Further, the present disclosure provides a host cell comprising any of the isolated nucleic acid or set of nucleic acids coding for the anti-Delta1 antibodies described herein.

Also provided herein is a method for producing the anti-Delta1 antibodies, comprising culturing the host cell described herein under suitable conditions allowing for expressing of the antibody, and harvesting the antibody thus produced from the cell culture (e.g., from the culture medium).

Further, the present disclosure provides a pharmaceutical composition, comprising any of the anti-Delta1 antibodies or a nucleic acid(s) encoding such, and a pharmaceutically acceptable carrier.

In yet another aspect, the present disclosure features a method of inhibiting activity and function of immunosuppressive γδ T cells, e.g. γδ1 T cells, in a subject, the method comprising administering to a subject in need thereof an effective amount of any of the anti-Delta1 antibodies disclosed herein or a pharmaceutical composition comprising such. In some embodiments, the subject in need thereof is a human patient having, suspected of having, or at risk for having a solid cancer. In some embodiments, the present disclosure features a method of treating a cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of any of the anti-Delta1 antibodies disclosed herein or a pharmaceutical composition comprising such. Exemplary solid tumors include, but are not limited to, pancreatic ductal adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer, glioblastoma, upper and lower gastrointestinal malignancies, squamous cell head and neck cancer, genitourinary cancer, ovarian cancer, endometrial cancer, renal cancer, bladder cancer, prostate cancer, neuroendocrine cancer, adrenocortical cancer, or sarcomas. In some examples, the effective amount of the pharmaceutical composition is sufficient to inhibit or block the activity and function of immunosuppressive γδ T cells, e.g., γδ1 T cells.

Any of the treatment methods described herein may further comprise administering to the subject an inhibitor of a checkpoint molecule, an activator of a co-stimulatory receptor, an inhibitor of an innate immune cell target, a chemotherapeutic agent, and/or any other anti-cancer treatment agent, including, but not limited to, a biologic, a small molecule inhibitor, and/or any form of radiation therapy, and/or cell based therapy. Examples of checkpoint molecules include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, TIGIT and VISTAA2aR. Examples of co-stimulatory receptors include, but are not limited to, OX40, GITR, CD137, CD40, CD27, and ICOS. Examples of innate immune cell targets include, but are not limited to, KIR, NKG2A, CD96, TLR, IDO, and galectin-9.

Also within the scope of the present disclosure are (i) pharmaceutical compositions for use in treating a disease associated with activation of immune suppressive γδ T cells (e.g., γδ1 T cells, wherein the pharmaceutical composition comprises any of the anti-Delta1 antibodies described herein or a nucleic acid(s) encoding such, and a pharmaceutically acceptable carrier; and (ii) uses of the anti-Delta1 antibodies or the encoding nucleic acids for manufacturing a medicament for use in treating the target diseases as described herein.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawing and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.

FIG. 1 includes a chart characterizing the binding specificity of phage-displayed anti-Delta-1 Fabs using phage ELISA.

FIGS. 2A-2C include charts showing epitope binning of Delta1-binding Fab clones (using γδ1 TCR containing the Delta-1C chain as the antigen immobilized in neutravidin-coated wells) using competitive phage ELISA. Phages displaying each of the Fab clones as indicated were incubated with purified Delta1-6 (FIG. 2A), Delta1-13 (FIG. 2B) or Delta1-17 (FIG. 2C) IgG that serves as the competitor.

FIGS. 3A-3F includes diagrams showing the binding affinity to γδ1 TCR containing the delta1C chain of Delta 1-1 (FIG. 3A), Delta 1-3 (FIG. 3B), Delta 1-6 (FIG. 3C), Delta1-10 (FIG. 3D), Delta1-13 (FIG. 3E), and Delta1-17 (FIG. 3F) as determined by surface plasmon resonance (SPR) analysis. Apparent K_(D) values are given in each diagram.

FIGS. 4A-4B includes diagrams showing the effects of Delta1-17 IgG on γδ T cell-mediated inhibition of T-cell activation.

FIG. 5 includes diagrams showing the thermostability of anti-Delta1 antibody clones as indicated (in IgG form). A thermal shift assay was performed and the curves are vertically offset for clarity.

FIGS. 6A-6H depict bar graphs showing Gamma chain binding of antibody clones Delta1-1 (FIG. 6A), Delta1-6 (FIG. 6B), Delta1-8 (FIG. 6D), Delta1-13 (FIG. 6E), Delta1-15 (FIG. 6F), Delta1-17 (FIG. 6G), and no phage control (FIG. 6H). by ELISA. Delta1C was expressed with a different Gamma chain (Gamma 3, 4, 5, 8 or 9) and purified as Fc-fusion proteins. These gamma-delta heterodimers were immobilized on the wells of a microtiter plate and probed with either clone Delta1-6, -10, -13, -17 or isotype. Delta1-8 and Delta1-17 show a pan-Gamma specificity.

FIG. 7A is a photograph of an immunohistochemical analysis, showing γδ T cells are enriched in human pancreatic cancer tissue compared to normal pancreas.

FIG. 7B is a bar graph showing that γδ T cells are enriched in tumors vs. peripheral blood.

FIGS. 8A and 8B are bar graphs showing that CD8+(FIG. 8A) and CD4+ T cells (FIG. 8B) abundantly infiltrate pancreatic tumors upon γδ T cell depletion and are responsible for mediating anti-tumor effects (as seen in Daley et al., 2016, Cell 166, 1485-1499).

FIGS. 9A, 9B, and 9C are bar graphs showing that depletion of γδ T cells enhances expression of relevant IO targets on αβ T cells. FIG. 9A: % of PD-1+ cells. FIG. 9B: % of CTLA4+ cells. FIG. 9C: % of OX40+ cells (as seen in Daley et al., 2016, Cell 166, 1485-1499).

FIG. 10 is a Kaplan-Meier plot showing significant extension of survival in animal models of pancreatic cancer (KPC mice) through γδ T cell depletion (TCRγβ−/−) and blocking (UC3-10A6 antibody; the antibody reacts with an epitope on the delta chain of the mouse Vγ2 TCR (V gamma 2 T cell receptor) (as seen in Daley et al., 2016, Cell 166, 1485-1499).

FIGS. 11A and 11B are bar graphs showing the results of in vitro culture assays with cells from a pancreatic cancer patient. FIG. 11A shows % TNF-α+ cells obtained upon culture of blood αβ T cells, alone or upon co-culture with intratumoral and blood γδ T cells. Bar 1: non-activated αβ T cell; Bar 2: activated αβ T cell; Bar 3: activated αβ T cell co-cultured with blood γδ T cells; Bar 4: activated αβ T cell co-cultured with tumor γδ T cells. FIG. 11B shows % TNF-α+ cells obtained upon treatment of a patient's tumor representative organoid with an anti-γδ antibody described herein (using Delta1-17 as an example) as compared to CD8 T cell activity.

FIGS. 12A-12C depict bar graphs showing co-culture assay of gamma delta (gdT) and alpha beta T cells (abT). Gamma delta cells are derived from tumor or blood, as indicated, alpha beta T cells are derived from blood of the same patient. FIG. 12A: Cells derived from colorectal cancer patient. FIGS. 12B and 12C: cells derived from two individual pancreatic cancer patients.

FIGS. 13A-13E depict bar graphs showing immune profile expression in a first colorectal cancer tumor sample (PDOTS) treated with isotype as compared to anti-delta1IgG1 antibody (using Delta1-17 IgG1 as an example); CD44 in CD3+ T cells (FIG. 13A), TNF-α in CD3+ T cells (FIG. 13B), TNF-α in CD4+ T cells (FIG. 13C), IFNγ in CD3+ T cells (FIG. 13D), IFNγ in CD4+ T cells (FIG. 13E).

FIGS. 14A-14F depict bar graphs showing immune profile expression in a second colorectal cancer tumor sample (PDOTS) treated with isotype as compared to anti-delta1IgG1 (using Delta1-17 as an example) or anti-PD1 antibodies or a combination thereof as indicated; CD44 in CD3+ T cells (FIG. 14A), CD44 in CD8+ T cells (FIG. 14B), IFNγ in CD3+ T cells (FIG. 14C), IFNγ in CD8+ T cells (FIG. 14D), TNF-α in CD3+ T cells (FIG. 14E), TNF-α in CD8+ T cells (FIG. 14F).

FIGS. 15A-15D depict bar graphs showing immune profile expression in a first colorectal cancer liver metastasis tumor sample (PDOTS) treated with isotype as compared to anti-Delta1 IgG1 antibody (using Delta1-17 IgG1 as an example); TNF-α in CD3+ T cells (FIG. 15A), TNF-α in CD8+ T cells (FIG. 15B), IFNγ in CD3+ T cells (FIG. 15C), IFNγ in CD8+ T cells (FIG. 15D).

FIGS. 16A-16D depict bar graphs showing immune profile expression in a second colorectal cancer liver metastasis tumor sample (PDOTS) treated with isotype as compared to anti-Delta1 IgG1 antibody (using Delta1-17 IgG1 as an example); TNF-α in CD3+ T cells (FIG. 16A), TNF-α in CD4+ T cells (FIG. 16B), IFNγ in CD3+ T cells (FIG. 16C), IFNγ in CD4+ T cells (FIG. 16D).

FIG. 17A-17C depict bar graphs showing immune profile expression in a first hepatocellular carcinoma tumor sample (PDOTS) treated with isotype as compared to anti-Delta1 IgG1 antibody (using Delta1-17 IgG1 as an example); CD44 in CD3+ T cells (FIG. 17A), TNF-α in CD3+ T cells (FIG. 17B), IFNγ in CD3+ T cells (FIG. 17C).

FIGS. 18A and 18B depict bar graphs showing immune profile expression in a second hepatocellular carcinoma tumor sample (PDOTS) treated with isotype as compared to anti-Delta1 IgG1 antibody (using Delta1-17 IgG1 as an example) or anti-PD1 antibody; CD44 in CD4+ T cells (FIG. 18A), TNF-α in CD4+ T cells (FIG. 18B).

FIGS. 19A and 19B depict bar graphs showing immune profile expression in a third hepatocellular carcinoma tumor sample (PDOTS) treated with isotype as compared to anti-Delta1 IgG1 antibody (using Delta1-17 IgG1 as an example); IFNγ in CD4+ T cells (FIG. 19A), TNF-α in CD8+ T cells (FIG. 19B).

FIGS. 20A and 20B depict graphs showing ADCC activity of Delta1-17 IgG1. A dose response of Delta1-17 IgG1 on ADCC, indicating the observed ADCC is dependent on Delta1-17 (FIG. 20A). ADCC activity of Delta1-17 IgG1 and IgG1 isotype in the presence and absence of coated γδ1 TCR sample, indicating that the observed ADCC activity is dependent on the presence of both Delta1-17 IgG1 and γδ1 TCR sample (FIG. 20B).

FIGS. 21A and 21B depict graphs showing ADCP activity of Delta1-17 IgG1. A dose response of Delta1-17 IgG1 on ADCP, indicating the observed ADCP is dependent on Delta1-17 (FIG. 21A). The observed ADCP is dependent on the specific interaction of Delta1-17 with its antigen (FIG. 21B).

DETAILED DESCRIPTION OF INVENTION

T cell receptors (TCRs) are disulfide-linked membrane-anchored heterodimeric proteins, expressed on the surface of T cells, where they recognize fragments of antigen presented by major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs) or other types of ligands presented on cell surface. Most T cells have TCRs comprising an alpha (α) and a beta (β) chain (known as αβ T cells), whereas a minority of T cells have TCRs made up of a gamma (γ) chain and a delta (δ) chain (known as γδ T cells).

The γδ TCRs recognize a variety of self and non-self antigens, such as small peptides, soluble or membrane proteins, phospholipids, prenyl pyrophosphates, and sulfatides. Due to their antigenic diversity, the γδ T cell can exert a wide range of different actions. For example, as γδ T cell activation does not require antigen processing and presentation by antigen-presenting cells (APCs), γδ T cells can be quickly activated and act during the early phase of immune responses. Similar to natural killer (NK) cells, γδ T cells also respond to stimulation by stress- and/or infection-induced ligands (Lafont et al., Front Immunol., 2014, 5: 622). Such ligands are typically weakly or not expressed under normal state, as they are up-regulated only in the presence of stress (DNA damage, heat stress) or infection. In addition, human γδ T cells also express pattern recognition receptors (PRR), such as Toll-like receptors (TLR), which modulate their activation (Shojaei et al., Cancer Res., 2009, 69(22): 8710-8717).

γδ T cells have been found to be both anti-tumorigenic and pro-tumorigenic (Lafont et al., Front Immunol., 2014, 5: 622). With respect to pancreatic ductal adenocarcinoma (PDA), γδ T cells have been found to make up a substantial fraction of the tumor-infiltrating T cells, where they have inhibitory functions on anti-cancer immunity mediated by αβ T cells. In a mouse model, the deletion, depletion, or blockade of γδ T cell recruitment was found to be protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβ T cells (Daley et al., Cell, 2016, 166: 1485-1499). In particular, the Delta1 subtype of γδ T cell receptors was found to be enriched among the tumor-infiltrating T cells.

Additionally, in a number of cancers, including melanoma, patients with higher frequencies of γδ1T cells had poorer overall survival. These patients with higher frequencies of γδ1T and lower frequencies of γδ2 T cells also have poorer outcome with anti-CTLA4 treatment. Moreover, in blood of glioblastoma multiforme patients compared with the healthy controls, the fraction of γδ1 T cells in peripheral blood mononuclear cells (PBMC) isolated from patients was significantly increased and the fraction of γδ2 in PBMCs was significantly decreased (Yue et al., 2018; Med Sci Monit, 2018; 24: 1784-1792). Functional test results showed that the immunosuppressive function of γδ1 T cells was enhanced and the killing function of γδ2 T cells was reduced.

Thus, antibodies specific to γδ T cells (e.g., specific to delta1 chain of a TCR comprising the delta1 chain and a gamma chain; “anti-Delta1 antibodies”) may be promising therapeutic agents for treating diseases associated with tumor-infiltrating γδ T cells (e.g., those in which tumor-infiltrating γδ T cells play an immunosuppressive role) or circulating γδ T cells, which may block conventional T cell activation and thus immune responses against pathologic cells (e.g., cancer cells). Without being bound by theory, an anti-Delta1 antibody may block the inhibitory function of the γδ T cells expressing such, thereby enhancing anti-tumor immune responses. Alternatively, or in addition, an anti-Delta1 antibody may also exert its therapeutic effect by inducing cytotoxicity, for example, ADCC, ADCP and/or CDC, against the target γδ T cells. A pathologic cell refers to a cell that contributes to the initiation and/or development of a disease, either directly or indirectly. In some embodiments, the anti-Delta1 antibody is an antibody drug conjugate, and exerts its effect by targeting a chemotherapeutic agent to the tumor site.

Accordingly, described herein are antibodies specific to γδ T cells (e.g., anti-Delta1 antibodies) and therapeutic uses thereof for rescuing inhibition of conventional T cell activity mediated by γδ T cells and/or treating diseases associated γδ T cell activation.

Antibodies Binding to the Delta1 Chain of γδ T Cells

The present disclosure provides antibodies that are specific to γδ T cells of a suitable species (e.g., human, mouse, rat, or a non-human primate such as monkey, chimpanzee, or ape), for example, specific to γδ1 T cells. Such antibodies may specifically bind a delta1 chain of the TCR expressed on γδ1 T cells. In some embodiments, the antibodies described herein bind the delta1 chain in a γδ1 heterodimer (e.g., a heterodimer of delta1 with any gamma chain known and/or described herein, such as gamma-9), which may be expressed on the surface of a γδ1 T cell.

The anti-Delta1 chain antibodies disclosed herein may exhibit one or more advantageous features, including, but not limited to, (i) high binding affinity to human delta 1 TCR (e.g., K_(D) lower than 10 nM, for example, clone Delta1-17); (ii) capable of binding to γδ1 TCRs containing various gamma chains, including gamma 3, 4, 5, 8, and/or 9, and others known in the art or described herein) (for example, clones Delta1-8, Delta1-10, Delta1-13, Delta-15, and Delta-17), optionally with a preference to gamma 9 (e.g., clones Delta1-1, Delta1-6, Delta1-10, and Delta1-13); (iii) high binding affinity to human delta 1 TCR, regardless of CDR1, CDR2 or CDR3 sequence, in particular, CDR3 sequence (iv) high specificity to human delta1 TCR (e.g., Delta1-17)) and/or (v) inhibiting γδ-T cell activation (e.g., Delta1-17) (vi) inhibition of immune suppressive function of γδ-T cell and/or promotion of effector cell recruitment and/or activation and activity of effector cells, and/or depletion of immunosuppressive γδ-T cells.

An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The term “antibody” is also meant to include so-called antibody mimetics. Antibody mimetics refers to small molecules, e.g., 3-30 kDa, which can be single amino acid chain molecules, which can specifically bind antigens but do not have an antibody-related structure. Antibody mimetics and their protein scaffolds, include, but are not limited to, Affibody molecules (Z domain of Protein A), Affilins (Gamma-B crystalline), Ubiquitin, Affimers (Cystatin). Affitins (Sac7d (from Sulfblobus acidocaldarius)), Alphabodies (Triple helix coiled coil). Anticalins (Lipocalins). Avimers (domains of various membrane receptors), DARPins (Ankyrin repeat motif), Fynomers (SH3 domain of Fyn), Kunitz domain peptides (Kunitz domains of various protease inhibitors), Ecallantide (Kalbitor), and Monobodies (fibronectin type III domain). Accordingly, some embodiments, the anti-Delta1 antibody is an antibody mimetic.

In some embodiments, the anti-Delta1 antibody is an antibody drug conjugate (ADC). ADCs generally comprise a monoclonal antibody against a target present on a cell, a cytotoxic drug, and a linker that attaches the antibody to the drug. Antibody-drug conjugates for cancer therapy are reviewed by Carter & Senter (2008), Cancer J. 14(3): 154-69, and Chari et al (2014) Angewandte Chemie International Edition 53: 3751. The cytotoxic moiety may be a polypeptide, which may be either directly or indirectly cytotoxic. When indirectly cytotoxic, the polypeptide may have enzymatic activity and can convert a relatively non-toxic prodrug into a cytotoxic drug (e.g., antibody-directed enzyme prodrug therapy; ADEPT). The cytotoxic moiety may comprise a drug selected from the group consisting of: a cytostatic agent (such as a taxane (e.g. docetaxel and, particularly, paclitaxel); an alkylating agent (such as cisplatin, carboplatin); an antimetabolite (such as 25 azathioprine, methotrexate); an antimitotic drug (such as vincristine); a topoisomerase inhibitor (such as doxorubicine, etoposide), etc. The cytotoxic moiety may be any known chemotherapeutic agent. The cytotoxic moiety may be an enterobacterial toxin, especially Pseudomonas exotoxin 20 A or calicheamicin. The cytotoxic moiety may also comprise a radioactive atom. The radioactive atom is typically selected from the group consisting of: iodine-123; iodine-125; iodine-131; indium-111; bromine-77; copper-67; arsenic-77; astatine-211; actinium-15 225; bismuth-212; bismuth-213; bismuth-217; lutetium-177; holmium-166; phosphorous-33; platinum-193; platinum-195; rhenium-186; rhenium-188; strontium-89; yttrium-90. gold-199, palladium-100; and antimony-211. In some embodiments, the cytotoxic moiety is capable of inhibiting at least one activity of cells expressing delta1 TCR. In some embodiments, the cytotoxic moiety is capable of inactivating or killing the cell. In order to facilitate the coupling between the cytotoxic moiety and the antibody, it is possible to directly conjugate the two agents, or to introduce a spacer molecule between them. Suitable spacers include poly(alkylene) glycols such as polyethylene glycol, and peptide linkers. Many suitable coupling techniques are well known in the art. Suitable agents allowing covalent, electrostatic or noncovalent binding of the moiety to the antibody include benzoquinone, carbodiimide and more particularly EDC (1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide hydrochloride), dimaleimide, dithiobis-nitrobenzoic acid (DTNB), N-succinimidyl S-acetyl thio-acetate (SATA), the bridging agents having one or more phenylazide groups reacting with the ultraviolets (U.V.) and preferably N-[-4-(azidosalicylamino)butyl]-3′-(2′-pyridyldithio)-propionamide (APDP), N-succinimid-yl 3-(2-25 pyridyldithio)propionate (SPDP), 6-hydrazino-nicotinamide (HYNIC). Another form of coupling, especially for the radioelements, includes the use of a bifunctional ion chelator. For example, chelates derived from EDTA or DTPA which have been developed for binding metals, especially radioactive metals, and immunoglobulins. Thus, DTPA and its derivatives can be substituted by different groups on the carbon chain in order to increase the stability and the rigidity of the ligand-metal complex, as is well known in the art.

In some embodiments, the anti-Delta1 antibody is conjugated to a drug to produce an antibody drug conjugate (ADC). In some embodiments, the anti-Delta1 antibody is an antibody drug conjugate (ADC). Suitable cytotoxic agents which can be conjugated to the anti-Delta1 antibody are described herein and known in the art. In some embodiments, the linker is cleavable. Non-limiting examples of linkers include, disulfide containing linkers that are cleavable through disulfide exchange, acid-labile linkers that are cleavable at acidic pH, and linkers that are cleavable by hydrolases (e.g., glycosyl hydrolases such as glucuronidases), esterases, and peptidases (e.g., peptide linkers and glucuronide linkers). In some embodiments, the linker is non-cleavable. In some embodiments, the drug is released via a proteolytic antibody degradation mechanism.

A typical antibody molecule comprises a heavy chain variable region (V_(H)) and a light chain variable region (V_(L)), which are usually involved in antigen binding. The V_(H) and V_(L) regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each V_(H) and V_(L) is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and/or the contact definition, all of which are well known in the art. See, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinforg.uk/abs).

The antibodies described herein are capable of binding to a T cell receptor delta1 polypeptide (anti-Delta1 antibody), which can be of a suitable source, for example, human or a non-human mammal (e.g., mouse, rat, rabbit, primate such as monkey, etc.).

The anti-Delta1 antibody as described herein may be a full-length antibody, which contains two heavy chains and two light chains, each including a variable domain and a constant domain. Alternatively, the anti-Delta1 antibody can be an antigen-binding fragment of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_(H) and C_(H)1 domains; (iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a single arm of an antibody (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of one or more V_(H) domain(s) (e.g., including but not limited to VHH domains (camelid or nanobodies); and (vi) an isolated complementarity determining region (CDR) that retains functionality. Furthermore, although the two domains of the Fv fragment, V_(L) and V_(H), are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_(L) and V_(H) regions pair to form monovalent molecules known as single chain Fv (scFv). See e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

In some embodiments, the anti-Delta1 antibodies described herein specifically bind to the corresponding target antigen or an epitope thereof, e.g., specifically binds a delta1 chain of a T cell γδ1 receptor. An antibody that “specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (e.g., a delta1 chain of a human TCR) or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens (e.g., a delta-2 chain of a human TCR) or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that “specifically binds” to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen, i.e., only baseline binding activity can be detected in a conventional method. Baseline binding activity refers to the binding activity detected in the conventional method when no antigen (blank control) or a different antigen (negative control) is used.

Specificity of the anti-Delta1 antibodies described herein can be measured using protein arrays, resulting in a specificity score (S score) as described herein and known in the art (see, e.g., Jeong et al., Mol Cell Proteomics. 2012 June; 11(6): O111.016253). Additionally, specificity of the anti-Delta1 antibodies described herein is assessed by comparing the K_(D) of binding of the anti-Delta1 antibodies to d1 with the K_(D) of binding to d2.

In some embodiments, the anti-Delta1 antibody described herein binds a motif that is common to TCR delta1 chains. In some embodiments, the anti-Delta1 antibody described herein binds to the Delta1 chain, regardless of CDR1, CDR2, or CDR3 sequences, e.g., regardless of CDR3 sequences. Sequences of TCR delta1 chains (e.g., human TCR delta1 chains, or TCR delta chains from other species) are well known in the art and can be found from publicly available databases, for example, the International Immunogenetics Information System® database (imgt.org), or GenBank. In some examples, the anti-Delta1 antibody may cross-react with different human delta1 chain sequences. Exemplary amino acid sequences of the extracellular regions (lacking transmembrane domain and cytoplasmic tail) of Delta1 chains (each of which differ in their CDR3 region) are provided below:

Human TCR: (SEQ ID NO: 26) AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIR QGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGVYAHSL IGGYRGGADKLIFGKGTRVTVEPRSQPHTKPSVFVMKNGTNVACLVKEFY PKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHD NKTVHSTDFEVKTDSTDHVKPKETENTKQPSKS Human TCR: (SEQ ID NO: 27) AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIR QGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGPRPSYS EELGDTHRADKLIFGKGTRVTVEPRSQPHTKPSVFVMKNGTNVACLVKEF YPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQH DNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKS Human TCR: (SEQ ID NO: 28) AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIR QGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEPNHFL NTDKLIFGKGTRVIVEPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRI NLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHS TDFEVKTDSTDHVKPKETENTKQPSKS

As used herein, “cross-react” means that an antibody shows binding activity (detectable by a conventional assay) to two or more different antigen sequences (e.g., human delta1 and delta2, or Delta1 chains with differing CDR regions, e.g., differing CDR3 regions). Such an antibody may have substantially similar binding affinity to these antigens, e.g., having a binding affinity to one antigen <10 fold (e.g., <5 fold or <2 fold) higher than that to another antigen as determined under the same assay conditions. Alternatively, such an antibody may have substantially higher binding affinity to one of these antigens as relative to another, for example, having a binding affinity to one antigen that is at least 10-fold higher (e.g., 20-fold higher, 50-fold higher, 100-fold higher, or 1,000-fold higher) than that to another antigen as determined under the same assay conditions.

In some embodiments, the anti-Delta1 antibody described herein preferentially binds a single human delta1 chain. In some embodiments, the antibody binds to a delta1 chain regardless of the sequence of the CDR regions, e.g., CDR3 region. In some embodiments, the anti-Delta1 antibody described herein preferentially binds a human delta1 chain as relative to another human delta-1 chain sequence. In some embodiments, the anti-Delta1 antibody described herein cross-reacts with more than a single human delta1 chain sequence. In some embodiments, the anti-Delta1 antibody preferentially binds human delta1 as relative to human delta-2 chain, a human delta-3 chain, a gamma chain (such as a gamma-9 chain), and/or a non-human delta1 chain. As used herein, an antibody “preferentially binds” a first antigen or an epitope thereof as compared with a second antigen or another epitope means that the antibody has a substantially higher binding affinity to the first antigen or the epitope thereof as relative to the second antigen or the other epitope, e.g., at least 10-fold higher (e.g., >20 fold, >50 fold, >100 fold, >1,000 fold or higher) as determined under the same assay conditions.

The anti-Delta1 antibody described herein may preferentially bind a human delta1 chain as relative to a non-human counterpart (e.g., a non-human primate delta1 chain), or vice versa. In other instances, the anti-Delta1 antibody described herein may cross-react to human and a non-human delta1 chain. For example, the antibody may cross-react to a human delta1 chain and a non-human primate delta1 chain.

In some instances, the anti-Delta1 antibody described herein does not bind to a human delta-2 chain, a human delta-3 chain, a gamma chain (such as a gamma-9 chain), and/or a non-human delta1 chain. In some embodiments, the anti-Delta1 antibody does not bind to a Delta-2 chain. (referred to herein as Delta2). In some embodiments, the anti-Delta1 antibody does not bind to a Delta-3 chain. (referred to herein as Delta3). In some embodiments, the anti-Delta1 antibody binds to neither a Delta-2 or Delta-3 chain. An antibody that does not bind an antigen means that no meaningful binding (e.g., only background binding or no binding at all) can be detected using a conventional assay (e.g., ELISA or surface plasmon resonance).

In some embodiments the antibody the anti-Delta1 antibody described herein binds to a T cell receptor comprising a delta1 chain and a gamma chain. Sequences of gamma chains are known in the art (see e.g., IMGT). Non-limiting examples of gamma chains include Gamma-1, gamma-2, gamma-3, gamma-4, gamma-5, gamma5P, gamma-8, gamma-9, gamma-10, gamma-11, gamma-a. Non-limiting examples of gamma chains are encoded by the following genes: •TRGV1, TRGV2, TRGV3, TRGV4, TRGV5, TRGV5P, TRGV8, TRGV9, TRGV10, TRGV11, and TRGVA. In some embodiments the antibody the anti-Delta1 antibody described herein binds to a T cell receptor comprising a delta1 chain and a gamma chain known in the art. In some embodiments, the anti-delta1 antibody described herein can bind to a TCR comprising any gamma chains. In some embodiments, the anti-delta1 antibody described herein can bind to a TCR comprising gamma3, 4, 5, 8 and 9 . . . The anti-Delta1 antibody as described herein preferably has a suitable binding affinity for the target antigen (e.g., a human delta1 chain) or antigenic epitopes thereof. As used herein, “binding affinity” refers to the apparent association constant or K_(A). The K_(A) is the reciprocal of the dissociation constant (K_(D)). The anti-Delta1 antibody described herein may have a binding affinity (K_(D)) of at least 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰ M, or lower for the target antigen or antigenic epitope. An increased binding affinity corresponds to a decreased K_(D). Higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher K_(A) (or a smaller numerical value K_(D)) for binding the first antigen than the K_(A) (or numerical value K_(D)) for binding the second antigen. In such cases, the antibody has specificity for the first antigen (e.g., a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold. In some embodiments, any of the anti-Delta1 antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation: [Bound]=[Free]/(Kd+[Free])

It is not always necessary to make an exact determination of K_(A), though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K_(A), and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

The anti-Delta1 antibodies described herein can inhibit γδ T cell activation, i.e., reducing the overall activity of γδ T cell, e.g., of an immune suppressive γδ T cell. Without being bound by theory, the anti-Delta1 antibodies described herein may inhibit the bioactivity of γδ1 T cells via directly blocking the activity of the γδ1 TCR expressed on the T cells. Alternatively or in addition, via binding to a γδ1 TCR, the anti-Delta1 antibodies may trigger cytotoxicity such as ADCC and/or ADCP and/or CDC, to eliminate T cells expressing the γδ1 TCR. Accordingly, the anti-Delta1 antibodies described herein may rescue immune suppression mediated by γδ T cells under, for example, cancer environment.

In some embodiments, the anti-Delta1 antibody as described herein inhibits the activity of γδ1 T cells. In some embodiments, the anti-Delta1 antibody as described herein inhibits the activity of γδ1 T cells by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein). In some embodiments, the inhibitory or suppressive potency of the anti-Delta1 antibody is measured by examining the ability of the antibody to “rescue” immune cells such as αβ T cells from the inhibitory activity of γδ T cells. In some embodiments, the anti-Delta1 antibody described herein may rescue the immune inhibition induced by γδ1 T cells. The anti-Delta1 antibody described herein may rescue the immune inhibition induced by γδ1 T cells by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater).

In some embodiments, the anti-Delta1 antibody as described herein activates or re-activates CD4+ helper cells and/or CD8+ effector cells in a tumor and/or in peripheral blood. In some embodiments, the anti-Delta1 antibody as described herein activates or re-activates CD4+ helper cells and/or CD8+ effector cells in a tumor and/or in peripheral blood by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein). In some embodiments, the ability of the anti-Delta1 antibody to activate CD4+ and/or CD8+ cells is measured by examining levels of proinflammatory cytokines. In some embodiments, the anti-Delta1 antibody is capable of increasing production of pro-inflammatory cytokines, including, but not limited to, IFNγ, TNF-α, and CD44, in the tumor. In some embodiments, the anti-Delta1 antibody is capable of increasing production of pro-inflammatory cytokines, including, but not limited to, IFNγ, TNF-α, and CD44, in the tumor by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the anti-Delta1 antibody as described herein can modulate, e.g., reduce, the ratio γδ1 T cells to γδ2 T cells. In some embodiments, the anti-Delta1 antibody as described herein modulates, e.g., reduces, the ratio γδ1 T cells to γδ2 T cells by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the anti-Delta1 antibody as described herein can modulate, e.g., reduce, the fraction γδ1 T cells present in PBMCs and/or the fraction of γδ1 T cells present in tumor localized immune cells. In some embodiments, the anti-Delta1 antibody as described herein modulates, e.g., reduces, the fraction γδ1 T cells present in PBMCs and/or the fraction of γδ1 T cells present in tumor localized immune cells by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the anti-Delta1 antibody as described herein can deplete a tumor of γδ T cells. In some embodiments, the anti-Delta1 antibody as described herein deplete a tumor of γδ T cells by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

The apparent inhibition constant (Ki^(app) or K_(i,app)), which provides a measure of inhibitor potency, is related to the concentration of inhibitor required to reduce enzyme activity and is not dependent on enzyme concentrations. The inhibitory activity of an anti-Delta1 antibody described herein can be determined by routine methods known in the art.

The K_(i,) ^(app) value of an antibody may be determined by measuring the inhibitory effect of different concentrations of the antibody on the extent of the reaction (e.g., enzyme activity); fitting the change in pseudo-first order rate constant (v) as a function of inhibitor concentration to the modified Morrison equation (Equation 1) yields an estimate of the apparent Ki value. For a competitive inhibitor, the Ki^(app) can be obtained from the y-intercept extracted from a linear regression analysis of a plot of k_(i,) ^(app) versus substrate concentration.

                                 (Equation  1) $v = {A \cdot \frac{\left( {\lbrack E\rbrack - \lbrack I\rbrack - K_{i}^{app}} \right) + {\sqrt{\left( {\lbrack E\rbrack - \lbrack I\rbrack - K_{i}^{app}} \right)^{2} + {4\lbrack E\rbrack}} \cdot K_{i}^{app}}}{2}}$

Where A is equivalent to v_(o)/E, the initial velocity (v_(o)) of the enzymatic reaction in the absence of inhibitor (I) divided by the total enzyme concentration (E).

In some embodiments, the anti-Delta1 antibody described herein may have a Ki^(app) value of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less for the target antigen or antigen epitope. In some embodiments, the anti-Delta1 antibody may have a lower Ki^(app) for a first target (e.g., a specific epitope of Delta1) relative to a second target (e.g., a different specific epitope of Delta1). Differences in Ki^(app) (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold. In some examples, the anti-Delta1 antibody inhibits a first antigen (e.g., a first protein in a first conformation or mimic thereof) better relative to a second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). In some embodiments, any of the anti-Delta1 antibodies may be further affinity matured to reduce the Ki^(app) of the antibody to the target antigen or antigenic epitope thereof.

The antibodies described herein can be murine, rat, human, or any other origin (including chimeric or humanized antibodies). Such antibodies are non-naturally occurring, i.e., would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof or isolated from antibody libraries).

Any of the antibodies described herein can be either monoclonal or polyclonal. A “monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.

Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, variable regions of V_(H) and V_(L) of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human V_(H) and V_(L) chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent V_(H) and V_(L) sequences as search queries. Human V_(H) and V_(L) acceptor genes are then selected.

The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions can be used to substitute for the corresponding residues in the human acceptor genes.

In another example, the antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications can be made in the variable region and/or the constant region.

A number of exemplary anti-Delta1 antibodies are provided below (CDR residues based on Kabat numbering are indicated by bolding):

Delta1-1 V_(H): (SEQ ID NO: 1) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAS ISSYYGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREE WMSYWYWPRYYYYGMDYWGQGTLVTVSS V_(L): (SEQ ID NO: 2) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQAALMSPITFG QGTKVEIKR Delta1-3 V_(H): (SEQ ID NO: 3) EVQLVESGGGLVQPGGSLRLSCAASGFTLSSSSIHWVRQAPGKGLEWVA SIYSSSGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR GLWSVWYYQFYSSMQGMDYWGQGTLVTVSS V_(L): (SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIY SASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGYEYPLTF GQGTKVEIKR Delta1-6 V_(H): (SEQ ID NO: 5) EVQLVESGGGLVQPGGSLRLSCAASGFTVYSSSIHWVRQAPGKGLEWVAS ISPYSGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARMW YLSGWWTGDALDYWGQGTLVTVSS V_(L): (SEQ ID NO: 6) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGYYSALITFG QGTKVEIKR Delta1-8 V_(H): (SEQ ID NO: 7) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAS IYSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARPY YWYPYYYWSGGWEYAAFDYWGQGTLVTVSS V_(L): (SEQ ID NO: 8) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGEYPITFGQ GTKVEIKR Delta1-9 V_(H): (SEQ ID NO: 9) EVQLVESGGGLVQPGGSLRLSCAASGFTVSYSSIHWVRQAPGKGLEWVAS ISPSSGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARQY WYYTFHYIYWLWALDYWGQGTLVTVSS V_(L): (SEQ ID NO: 10) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSQFSGPITFG QGTKVEIKR Delta1-10 V_(H): (SEQ ID NO: 11) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSSSIHWVRQAPGKGLEWVAS ISSSSGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSM VWYWGLNGYEEYAGGMDYWGQGTLVTVSS V_(L): (SEQ ID NO: 12) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQ GTKVEIKR Delta1-12 V_(H): (SEQ ID NO: 13) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAS IYSYYGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARTS KYYYVYEYYYHMHIAMDYWGQGTLVTVSS V_(L): (SEQ ID NO: 14) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSNHSTLITF GQGTKVEIKR Delta1-13 V_(H): (SEQ ID NO: 15) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSSSIHWVRQAPGKGLEWVAS ISSSSGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARTT SYIDEYFGFGWYAMDYWGQGTLVTVSS V_(L): (SEQ ID NO: 12) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQ GTKVEIKR Delta1-14 V_(H): (SEQ ID NO: 17) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSSSIHWVRQAPGKGLEWVAS IHSSSSSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVY WPYQYGPWAGFDYWGQGTLVTVSS V_(L): (SEQ ID NO: 18) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSYWYPITFGQ GTKVEIKR Delta1-15 V_(H): (SEQ ID NO: 19) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSSSIHWVRQAPGKGLEWVAS ISSSSGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYS WIYDSWWSGWAMDYWGQGTLVTVSS V_(L): (SEQ ID NO: 12) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQ GTKVEIKR Delta1-16 V_(H): (SEQ ID NO: 21) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAS ISSSSGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYW HGWHFGHYGYTWALDYWGQGTLVTVSS V_(L): (SEQ ID NO: 12) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQ GTKVEIKR Delta1-17 V_(H): (SEQ ID NO: 23) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAS IYSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARDP GMYYWYYSGSAYEGYGLDYWGQGTLVTVSS V_(L): (SEQ ID NO: 24) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSGDDLITFGQ GTKVEIKR

A number of exemplary anti-Delta1 antibodies CDRs (based on Kabat numbering) are provided below.

TABLE 1 Selected Antibody CDR Sequences SEQ ID Clone Sequence NO: Delta1-1 V_(H) CDR1 FTVSSSSIH 43 V_(H) CDR2 SISSYYGYTSYADSVKG 44 V_(H) CDR3 EEWMSYWYWPRYYYYGMDY 45 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQAALMSPIT 48 Delta1-3 V_(H) CDR1 FTLSSSSIH 49 V_(H) CDR2 SIYSSSGYTSYADSVKG 50 V_(H) CDR3 GLWSVWYYQFYSSMQGMDY 51 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQGYEYPLT 52 Delta1-6 V_(H) CDR1 FTVYSSSIH 53 V_(H) CDR2 SISPYSGSTSYADSVKG 54 V_(H) CDR3 MWYLSGWWTGDALDY 55 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQGYYSALIT 56 Delta1-8 V_(H) CDR1 FTVSSSSIH 43 V_(H) CDR2 SIYSSSGYTYYADSVKG 57 V_(H) CDR3 PYYWYPYYYWSGGWEYAAFDY 58 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQYGEYPIT 59 Delta1-9 V_(H) CDR1 FTVSYSSIH 60 V_(H) CDR2 SISPSSGYTSYADSVKG 61 V_(H) CDR3 QYWYYTFHYIYWLWALDY 62 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQSQFSGPIT 63 Delta1-10 V_(H) CDR1 FTFSSSSIH 64 V_(H) CDR2 SISSSSGSTSYADSVKG 65 V_(H) CDR3 SMVWYWGLNGYEEYAGGMDY 66 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQSSSSLIT 67 Delta1-12 V_(H) CDR1 FTVSSSSIH 43 V_(H) CDR2 SIYSYYGYTYYADSVKG 68 V_(H) CDR3 TSKYYYVYEYYYHMIIIAMDY 69 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQSSNHSTLIT 70 Delta1-13 V_(H) CDR1 FTFSSSSIH 64 V_(H) CDR2 SISSSSGSTSYADSVKG 65 V_(H) CDR3 TTSYIDEYFGFGWYAMD 71 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQSSSSLIT 67 Delta1-14 V_(H) CDR1 FTFSSSSIH 64 V_(H) CDR2 SIRSSSSSTYYADSVKG 72 V_(H) CDR3 VYWPYQYGPWAGFDY 73 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQSYWYPIT 74 Delta 1-15 V_(H) CDR1 FTFSSSSIH 64 V_(H) CDR2 SISSSSGSTSYADSVKG 65 V_(H) CDR3 YSWIYDSWWSGWAMDY 75 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQSSSSLIT 67 Delta1-16 V_(H) CDR1 FTVSSSSIH 43 V_(H) CDR2 SISSSSGSTSYADSVKG 65 V_(H) CDR3 YWHGWHFGHYGYTWALDY 76 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQSSSSLIT 67 Delta1-17 V_(H) CDR1 FTVSSSSIH 43 V_(H) CDR2 SIYSSSGYTYYADSVKG 57 V_(H) CDR3 DPGMYYWYYSGSAYEGYGLDY 77 V_(L) CDR1 RASQSVSSAVA 46 V_(L) CDR2 SASSLYS 47 V_(L) CDR3 QQSGDDLIT 78

In some embodiments, the anti-Delta1 antibodies described herein bind to the same epitope as any of the exemplary antibodies listed above or competes against the exemplary antibody from binding to the delta1 chain. An “epitope” refers to the site on a target antigen that is recognized and bound by an antibody. The site can be entirely composed of amino acid components, entirely composed of chemical modifications of amino acids of the protein (e.g., glycosyl moieties), or composed of combinations thereof. Overlapping epitopes include at least one common amino acid residue. An epitope can be linear, which is typically 6-15 amino acids in length. Alternatively, the epitope can be conformational. The epitope to which an antibody binds can be determined by routine technology, for example, the epitope mapping method (see, e.g., descriptions below). An antibody that binds the same epitope as an exemplary antibody described herein may bind to exactly the same epitope or a substantially overlapping epitope (e.g., containing less than 3 non-overlapping amino acid residue, less than 2 non-overlapping amino acid residues, or only 1 non-overlapping amino acid residue) as the exemplary antibody. Whether two antibodies compete against each other from binding to the cognate antigen can be determined by a competition assay, which is well known in the art.

In some examples, the anti-Delta1 antibody described herein comprises the same V_(H) and/or V_(L) CDRs as an exemplary antibody listed above. Two antibodies having the same V_(H) and/or V_(L) CDRs means that their CDRs are identical when determined by the same approach (e.g., the Kabat approach or the Chothia approach as known in the art). Such anti-Delta1 antibodies may have the same V_(H), the same V_(L), or both as compared to an exemplary antibody described herein.

Also within the scope of the present disclosure are functional variants of any of the exemplary anti-Delta1 antibodies as disclosed herein. Such functional variants are substantially similar to the exemplary antibody, both structurally and functionally. A functional variant comprises substantially the same V_(H) and/or VL CDRs as the exemplary antibody. For example, it may comprise only up to 10 (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid residue variations in the total CDR regions of the antibody and binds the same epitope of Delta1 with substantially similar affinity (e.g., having a K_(D) value in the same order). Alternatively or in addition, the amino acid residue variations are conservative amino acid residue substitutions. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the anti-Delta1 antibody may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRs of an exemplary antibody described herein. Alternatively or in addition, the anti-Delta1 antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the V_(L) CDRs as an exemplary antibody described herein. “Individually” means that one particular heavy chain or light chain CDR of an antibody shares the described sequence identify to the corresponding heavy chain or light chain CDR of an exemplary antibody, e.g., disclosed herein. “Collectively” means that the three heavy chain or light chain CDRs of an antibody, in combination, share the described sequence identity to the three corresponding heavy chain or light chain CDRs of an exemplary antibody, e.g., those described herein.

In some embodiments, the anti-Delta1 antibody described herein may comprise a V_(H) that has at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity to the V_(H) of an exemplary antibody described herein. Alternatively or in addition, the anti-Delta1 antibody may comprise a V_(L) that has at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity to the V_(L) of an exemplary antibody described herein.

The “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46. In some embodiments, the anti-Delta1 antibody comprises a VL CDR2 having the sequence of SEQ ID NO: 47. In some embodiments, the anti-Delta1 antibody comprises a VL CDR3 having a sequence selected from SEQ ID NOs: 48, 52, 56, 59, 63, 67, 70, 74 and 78. In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, and a VL CDR3 having a sequence selected from SEQ ID NOs: 48, 52, 56, 59, 63, 67, 70, 74 and 78.

In some embodiments, anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, VL CDR2 having the sequence of SEQ ID NO: 47, and VL CDR3 having the sequence of SEQ ID NO: 48. In some embodiments, anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, VL CDR2 having the sequence of SEQ ID NO: 47, and VL CDR3 having the sequence of SEQ ID NO: 52. In some embodiments, anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, VL CDR2 having the sequence of SEQ ID NO: 47, and VL CDR3 having the sequence of SEQ ID NO: 56. In some embodiments, anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, VL CDR2 having the sequence of SEQ ID NO: 47, and VL CDR3 having the sequence of SEQ ID NO: 59. In some embodiments, anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, VL CDR2 having the sequence of SEQ ID NO: 47, and VL CDR3 having the sequence of SEQ ID NO: 63. In some embodiments, anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, VL CDR2 having the sequence of SEQ ID NO: 47, and VL CDR3 having the sequence of SEQ ID NO: 67. In some embodiments, anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, VL CDR2 having the sequence of SEQ ID NO: 47, and VL CDR3 having the sequence of SEQ ID NO: 70. In some embodiments, anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, VL CDR2 having the sequence of SEQ ID NO: 47, and VL CDR3 having the sequence of SEQ ID NO: 74. In some embodiments, anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, VL CDR2 having the sequence of SEQ ID NO: 47, and VL CDR3 having the sequence of SEQ ID NO: 78.

In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having a sequence selected from SEQ ID NOs: 43, 49, 53, 60, and 64. In some embodiments, the anti-Delta1 antibody comprises a VH CDR2 having a sequence selected from SEQ ID NOs: 44, 50, 54, 57, 61, 65, 68, and 72. In some embodiments, the anti-Delta1 antibody comprises a VH CDR3 having a sequence selected from SEQ ID NOs: 45, 51, 55, 58, 62, 66, 69, 71, 73, 75, 76, and 77. In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having a sequence selected from SEQ ID NOs: 43, 49, 53, 60, and 64, a VH CDR2 having a sequence selected from SEQ ID NOs: 44, 50, 54, 57, 61, 65, 68, and 72 and a VH CDR3 having a sequence selected from SEQ ID NOs: 45, 51, 55, 58, 62, 66, 69, 71, 73, 75, 76, and 77.

In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 43. In some embodiments, the anti-Delta1 antibody comprises a VH CDR2 having a sequence selected from SEQ ID NOs: 44, 57, 68 and 65. In some embodiments, the anti-Delta1 antibody comprises a VH CDR3 having a sequence selected from SEQ ID NOs: 45, 58, 69, 76, and 77. In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 43, a VH CDR2 having a sequence selected from SEQ ID NOs: 44, 57, 68 and 65, and a VH CDR3 having a sequence selected from SEQ ID NOs: 45, 58, 69, 76, and 77. In some embodiments, anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 43, VH CDR2 having the sequence of SEQ ID NO: 44, and VH CDR3 having the sequence of SEQ ID NO: 45. In some embodiments, anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 43, VH CDR2 having the sequence of SEQ ID NO: 57, and VH CDR3 having the sequence of SEQ ID NO: 58. In some embodiments, anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 43, VH CDR2 having the sequence of SEQ ID NO: 68, and VH CDR3 having the sequence of SEQ ID NO: 69. In some embodiments, anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 43, VH CDR2 having the sequence of SEQ ID NO: 65, and VH CDR3 having the sequence of SEQ ID NO: 76. In some embodiments, anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 43, VH CDR2 having the sequence of SEQ ID NO: 57, and VH CDR3 having the sequence of SEQ ID NO: 77.

In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 49. In some embodiments, the anti-Delta1 antibody comprises a VH CDR2 having the sequence of SEQ ID NO: 50. In some embodiments, the anti-Delta1 antibody comprises a VH CDR3 having the sequence of SEQ ID NO: 51. In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 49, a VH CDR2 having the sequence of SEQ ID NO: 50, and a VH CDR3 having the sequence of SEQ ID NOs: 51.

In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 53. In some embodiments, the anti-Delta1 antibody comprises a VH CDR2 having the sequence of SEQ ID NO: 54. In some embodiments, the anti-Delta1 antibody comprises a VH CDR3 having the sequence of SEQ ID NO: 55. In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 53, a VH CDR2 having the sequence of SEQ ID NO: 54, and a VH CDR3 having the sequence of SEQ ID NOs: 55.

In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 60. In some embodiments, the anti-Delta1 antibody comprises a VH CDR2 having the sequence of SEQ ID NO: 61. In some embodiments, the anti-Delta1 antibody comprises a VH CDR3 having the sequence of SEQ ID NO: 62. In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 60, a VH CDR2 having the sequence of SEQ ID NO: 61, and a VH CDR3 having the sequence of SEQ ID NOs: 62.

In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 64. In some embodiments, the anti-Delta1 antibody comprises a VH CDR2 having a sequence selected from SEQ ID NOs: 65 and 72. In some embodiments, the anti-Delta1 antibody comprises a VH CDR3 having a sequence selected from SEQ ID NOs: 66, 71, 73, and 75. In some embodiments, the anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 64, a VH CDR2 having a sequence selected from SEQ ID NOs: 65 and 72, and a VH CDR3 having a sequence selected from SEQ ID NOs: 66, 71, 73, and 75. In some embodiments, anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 64, VH CDR2 having the sequence of SEQ ID NO: 65, and VH CDR3 having the sequence of SEQ ID NO: 66. In some embodiments, anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 64, VH CDR2 having the sequence of SEQ ID NO: 65, and VH CDR3 having the sequence of SEQ ID NO: 71. In some embodiments, anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 64, VH CDR2 having the sequence of SEQ ID NO: 72, and VH CDR3 having the sequence of SEQ ID NO: 73. In some embodiments, anti-Delta1 antibody comprises a VH CDR1 having the sequence of SEQ ID NO: 64, VH CDR2 having the sequence of SEQ ID NO: 65, and VH CDR3 having the sequence of SEQ ID NO: 75

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having a sequence selected from SEQ ID NOs: 48, 52, 56, 59, 63, 67, 70, 74 and 78, a VH CDR1 having a sequence selected from SEQ ID NOs: 43, 49, 53, 60, and 64, a VH CDR2 having a sequence selected from SEQ ID NOs: 44, 50, 54, 57, 61, 65, 68, and 72, and a VH CDR3 having a sequence selected from SEQ ID NOs: 45, 51, 55, 58, 62, 66, 69, 71, 73, 75, 76, and 77.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 48, a VH CDR1 having the sequence of SEQ ID NO: 43, a VH CDR2 having the sequence of SEQ ID NO: 44, and a VH CDR3 having the sequence of SEQ ID NOs: 45.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 52, a VH CDR1 having the sequence of SEQ ID NO: 49, a VH CDR2 having the sequence of SEQ ID NO: 50, and a VH CDR3 having the sequence of SEQ ID NOs: 51.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 56, a VH CDR1 having the sequence of SEQ ID NO: 53, a VH CDR2 having the sequence of SEQ ID NO: 54, and a VH CDR3 having the sequence of SEQ ID NOs: 55.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 59, a VH CDR1 having the sequence of SEQ ID NO: 43, a VH CDR2 having the sequence of SEQ ID NO: 57, and a VH CDR3 having the sequence of SEQ ID NOs: 58.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 63, a VH CDR1 having the sequence of SEQ ID NO: 60, a VH CDR2 having the sequence of SEQ ID NO: 61, and a VH CDR3 having the sequence of SEQ ID NOs: 62.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 67, a VH CDR1 having the sequence of SEQ ID NO: 64, a VH CDR2 having the sequence of SEQ ID NO: 65, and a VH CDR3 having the sequence of SEQ ID NOs: 66.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 70, a VH CDR1 having the sequence of SEQ ID NO: 43, a VH CDR2 having the sequence of SEQ ID NO: 68, and a VH CDR3 having the sequence of SEQ ID NOs: 69.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 67, a VH CDR1 having the sequence of SEQ ID NO: 64, a VH CDR2 having the sequence of SEQ ID NO: 65, and a VH CDR3 having the sequence of SEQ ID NOs: 71.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 74, a VH CDR1 having the sequence of SEQ ID NO: 64, a VH CDR2 having the sequence of SEQ ID NO: 72, and a VH CDR3 having the sequence of SEQ ID NOs: 73.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 67, a VH CDR1 having the sequence of SEQ ID NO: 64, a VH CDR2 having the sequence of SEQ ID NO: 65, and a VH CDR3 having the sequence of SEQ ID NOs: 75.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 67, a VH CDR1 having the sequence of SEQ ID NO: 43, a VH CDR2 having the sequence of SEQ ID NO: 65, and a VH CDR3 having the sequence of SEQ ID NOs: 76.

In some embodiments, the anti-Delta1 antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 46, a VL CDR2 having the sequence of SEQ ID NO: 47, a VL CDR3 having the sequence of SEQ ID NOs: 78, a VH CDR1 having the sequence of SEQ ID NO: 43, a VH CDR2 having the sequence of SEQ ID NO: 57, and a VH CDR3 having the sequence of SEQ ID NOs: 77.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of the VL region of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of the VH region of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17. In some embodiments, the anti-Delta1 antibody comprises a VL region and a VH region having sequences of the VL and VH regions of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17.

In some embodiments, the anti-Delta1 antibody comprises a VL region having a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24. In some embodiments, the anti-Delta1 antibody comprises a VH region having a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the anti-Delta1 antibody comprises a VL region a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24 and a VH region having a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of the VL region of Delta1-17. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of VH region of Delta1-17. In some embodiments, the anti-Delta1 antibody comprises a VL region and a VH region having the sequence of the VL and the VH of Delta1-17.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 2. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 1. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 2 and a VH region having the sequence of SEQ ID NO: 1.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 4. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 3. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 4 and a VH region having the sequence of SEQ ID NO: 3.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 6. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 5. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 6 and a VH region having the sequence of SEQ ID NO: 5.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 8. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 7. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 8 and a VH region having the sequence of SEQ ID NO: 7.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 10. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 9. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 10 and a VH region having the sequence of SEQ ID NO: 9.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 12. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 11. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 15. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 19. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 21. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 12 and a VH region having the sequence of SEQ ID NO: 11. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 12 and a VH region having the sequence of SEQ ID NO: 15. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 12 and a VH region having the sequence of SEQ ID NO: 19. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 12 and a VH region having the sequence of SEQ ID NO: 21.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 14. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 13. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 14 and a VH region having the sequence of SEQ ID NO: 13.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 18. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 17. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 18 and a VH region having the sequence of SEQ ID NO: 17.

In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 24. In some embodiments, the anti-Delta1 antibody comprises a VH region having the sequence of SEQ ID NO: 23. In some embodiments, the anti-Delta1 antibody comprises a VL region having the sequence of SEQ ID NO: 24 and a VH region having the sequence of SEQ ID NO: 23.

In some embodiments, the anti-Delta1 antibody has a VL sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(L) sequence of of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(H) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17. In some embodiments, the anti-Delta1 antibody has a VL sequence comprising the V_(L) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising the V_(H) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of or consisting of the V_(L) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of the V_(H) sequence of or consisting of the V_(H) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(L) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(H) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence comprising the V_(L) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 and a V_(H) sequence comprising the V_(H) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of the V_(L) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 and a V_(H) sequence consisting essentially of the V_(H) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence consisting of the V_(L) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17 and a V_(H) sequence consisting of the V_(H) sequence of an antibody selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, and Delta1-17.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(L) sequence of Delta1-17. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(H) sequence of Delta1-17. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising the V_(L) sequence of Delta1-17. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising the V_(H) sequence of Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of the V_(L) sequence of Delta1-17 or consisting of the V_(L) sequence of Delta1-17. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of the V_(H) sequence of Delta1-17 or consisting of the V_(H) sequence of Delta1-17.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(L) sequence of Delta1-17 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(H) sequence of Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence comprising the V_(L) sequence of Delta1-17 and a V_(H) sequence comprising the V_(H) sequence of Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of the V_(L) sequence of Delta1-17 and a V_(H) sequence consisting essentially of the V_(H) sequence of Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence consisting of the V_(L) sequence of Delta1-17 and a V_(H) sequence consisting of the V_(H) sequence of Delta1-17.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18, and 24. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of or consisting of a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of or consisting of a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 8′7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the isolated antibody has a V_(L) sequence comprising a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24 and a V_(H) sequence comprising a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24 and a V_(H) sequence consisting essentially of a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting of a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 18 and 24 and a V_(H) sequence consisting of a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 2. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising SEQ ID NO: 2. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 1. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 2 or consisting of SEQ ID NO: 2. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 1 or consisting of SEQ ID NO: 1.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 2 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 2 and a V_(H) sequence comprising SEQ ID NO: 1. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 2 and a V_(H) sequence consisting essentially of SEQ ID NO: 1. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 2 and a V_(H) sequence consisting of SEQ ID NO: 1.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 3. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising SEQ ID NO: 4. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 3. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 4 or consisting of SEQ ID NO: 4. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 3 or consisting of SEQ ID NO: 3.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 3. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 4 and a V_(H) sequence comprising SEQ ID NO: 3. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 4 and a V_(H) sequence consisting essentially of SEQ ID NO: 3. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 4 and a V_(H) sequence consisting of SEQ ID NO: 3.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 6. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 5. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising SEQ ID NO: 6. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 5. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 6 or consisting of SEQ ID NO: 6. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 5 or consisting of SEQ ID NO: 5.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 6 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 5. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 6 and a V_(H) sequence comprising SEQ ID NO: 5. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 6 and a V_(H) sequence consisting essentially of SEQ ID NO: 5. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 6 and a V_(H) sequence consisting of SEQ ID NO: 5.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 7. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising SEQ ID NO: 8. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 7. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 8 or consisting of SEQ ID NO: 8. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 7 or consisting of SEQ ID NO: 7.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 7. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 8 and a V_(H) sequence comprising SEQ ID NO: 7. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 8 and a V_(H) sequence consisting essentially of SEQ ID NO: 7. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 8 and a V_(H) sequence consisting of SEQ ID NO: 7.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 10. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 9. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising SEQ ID NO: 10. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 9. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 10 or consisting of SEQ ID NO: 10. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 9 or consisting of SEQ ID NO: 9.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 10 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 9. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 10 and a V_(H) sequence comprising SEQ ID NO: 9. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 10 and a V_(H) sequence consisting essentially of SEQ ID NO: 9. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 10 and a V_(H) sequence consisting of SEQ ID NO: 9.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 12. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 11. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 15. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 19. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 21. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising SEQ ID NO: 12. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 11. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 15. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 19. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 21. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 12 or consisting of SEQ ID NO: 12. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 11 or consisting of SEQ ID NO: 11. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 15 or consisting of SEQ ID NO: 15. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 19 or consisting of SEQ ID NO: 19. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 21 or consisting of SEQ ID NO: 21.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 12 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 11. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 12 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 15. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 12 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 19. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 12 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 21. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 12 and a V_(H) sequence comprising SEQ ID NO: 11. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 12 and a V_(H) sequence comprising SEQ ID NO: 15. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 12 and a V_(H) sequence comprising SEQ ID NO: 19. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 12 and a V_(H) sequence comprising SEQ ID NO: 21. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 12 and a V_(H) sequence consisting essentially of SEQ ID NO: 11. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 12 and a V_(H) sequence consisting essentially of SEQ ID NO: 15. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 12 and a V_(H) sequence consisting essentially of SEQ ID NO: 19. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 12 and a V_(H) sequence consisting essentially of SEQ ID NO: 21. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 12 and a V_(H) sequence consisting of SEQ ID NO: 11. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 12 and a V_(H) sequence consisting of SEQ ID NO: 15. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 12 and a V_(H) sequence consisting of SEQ ID NO: 19. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 12 and a V_(H) sequence consisting of SEQ ID NO: 21.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 14. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 13. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising SEQ ID NO: 14. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 13.

In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 14 or consisting of SEQ ID NO: 14. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 13 or consisting of SEQ ID NO: 13.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 14 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 13. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 14 and a V_(H) sequence comprising SEQ ID NO: 13. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 14 and a V_(H) sequence consisting essentially of SEQ ID NO: 13. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 14 and a V_(H) sequence consisting of SEQ ID NO: 13.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 18. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 17. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising SEQ ID NO: 18. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 17. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 18 or consisting of SEQ ID NO: 18. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of SEQ ID NO: 17 or consisting of SEQ ID NO: 17.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 18 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 17. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 18 and a V_(H) sequence comprising SEQ ID NO: 17. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 18 and a V_(H) sequence consisting essentially of SEQ ID NO: 17. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 18 and a V_(H) sequence consisting of SEQ ID NO: 17.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 24. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 23. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising SEQ ID NO: 24. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising SEQ ID NO: 23.

In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of or consisting of SEQ ID NO: 24. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of or consisting of SEQ ID NO: 23.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 24 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 23. In some embodiments, the isolated antibody has a V_(L) sequence comprising SEQ ID NO: 24 and a V_(H) sequence comprising SEQ ID NO: 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of SEQ ID NO: 24 and a V_(H) sequence consisting essentially of SEQ ID NO: 23. In some embodiments, the isolated antibody has a V_(L) sequence consisting of SEQ ID NO: 24 and a V_(H) sequence consisting of SEQ ID NO: 23.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(L) sequence of Delta1-17. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(H) sequence of Delta1-17. In some embodiments, the anti-Delta1 antibody has a V_(L) sequence comprising the V_(L) sequence of Delta1-17. In some embodiments, the anti-Delta1 antibody has a V_(H) sequence comprising the V_(H) sequence of Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of the V_(L) sequence of Delta1-17 or consisting of the V_(L) sequence of Delta1-17. In some embodiments, the isolated antibody has a V_(H) sequence consisting essentially of the V_(H) sequence of Delta1-17 or consisting of the V_(H) sequence of Delta1-17.

In some embodiments, the anti-Delta1 antibody has a V_(L) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(L) sequence of Delta1-17 and has a V_(H) sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the V_(H) sequence of Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence comprising the V_(L) sequence of Delta1-17 and a V_(H) sequence comprising the V_(H) sequence of Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence consisting essentially of the V_(L) sequence of Delta1-17 and a V_(H) sequence consisting essentially of the V_(H) sequence of Delta1-17. In some embodiments, the isolated antibody has a V_(L) sequence consisting of the V_(L) sequence of Delta1-17 and a V_(H) sequence consisting of the V_(H) sequence of Delta1-17.

In some embodiments, the heavy chain of any of the anti-Delta1 antibodies as described herein may further comprise a heavy chain constant region (C_(H)) or a portion thereof (e.g., C_(H)1, C_(H)2, C_(H)3, or a combination thereof). The heavy chain constant region can of any suitable origin, e.g., human, mouse, rat, or rabbit. In one specific example, the heavy chain constant region is from a human IgG (a gamma heavy chain) of any IgG subfamily as described herein. (for example, human IgG1, IgG1 LALA, IgG4, or human IgG4 Fab Arm Exchange mutant). In one example, the constant region is from human IgG1, an exemplary amino acid sequence of which is heavy chain constant regions are provided below (SEQ ID NO: 31). In some embodiments, the heavy chain constant region of the anti-Delta1 antibodies described herein may comprise a single domain (e.g., C_(H)1, C_(H)2, or C_(H)3) or a combination of any of the single domains, of a constant region (e.g., SEQ ID NO: 80-82). In some embodiments, the light chain constant region of the antibodies described herein may comprise a single domain (e.g., CL), of a constant region (e.g., SEQ ID NO: 79). Exemplary light and heavy chain sequences are listed below. The hIgG1 LALA sequence includes two mutations, L234A and L235A, which suppress FcgR binding as well as a P329G mutation to abolish complement C1q binding, thus abolishing all immune effector functions. The hIgG4 Fab Arm Exchange Mutant sequence includes a mutation to suppress Fab Arm Exchange (S228P). For manufacturing purposes, it may be desirable to delete the C-terminal lysine (“K”) residue in the heavy chain sequences below. Therefore, in some embodiments, the C-terminal lysine (“K”) residue may not be present in each of the heavy chain sequences presented below.

IgK Light Chain (LC) (SEQ ID NO: 79): TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC IgG1 HC (SEQ ID NO: 31): ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK IgG1 Heavy Chain (HC) LALA (SEQ ID NO: 80): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG4 HC (SEQ ID NO: 81): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLK IgG4 HC Fab Arm Exchange mutant (SEQ ID NO: 82) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK

In any of the embodiments described herein, an anti-Delta1 antibody of the disclosure may comprise a single domain of a constant region having the sequence of SEQ ID NO: 79. In any of the embodiments described herein, an anti-Delta1 antibody of the disclosure may comprise a light chain constant region having the sequence of SEQ ID NO: 79. In any of the embodiments described herein, an anti-Delta1 antibody of the disclosure may comprise the heavy chain constant region having the sequence of SEQ ID NO: 31. In any of the embodiments described herein, an anti-Delta1 antibody of the disclosure may comprise the heavy chain constant region having the sequence of SEQ ID NO: 80. In any of the embodiments described herein, an anti-Delta1 antibody of the disclosure may comprise the heavy chain constant region having the sequence of SEQ ID NO: 81. In any of the embodiments described herein, an anti-Delta1 antibody of the disclosure may comprise the heavy chain constant region having the sequence of SEQ ID NO: 82.

A non-limiting example of such sequences are as follows:

Delta1-17 IgK Light Chain (SEQ ID NO: 83): DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYS ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSGDDLITFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVIEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Delta1-17 IgG1 HC (SEQ ID NO: 84): EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAS IYSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARDP GMYYWYYSGSAYEGYGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Delta1-17 IgG1 HC LALA (SEQ ID NO: 85): EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAS IYSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARDP GMYYWYYSGSAYEGYGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Delta1-17 IgG4 HC (SEQ ID NO: 86): EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAS IYSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARDP GMYYWYYSGSAYEGYGLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTIPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK Delta1-17 IgG4 HC Fab Arm Exchange mutant (SEQ ID NO: 87): EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAS IYSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARDP GMYYWYYSGSAYEGYGLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK

In some embodiments, the anti-Delta1-17 antibody described herein may comprise a single domain (e.g., CL), of a constant region having the sequence of SEQ ID NO: 79. In some embodiments, anti-Delta1-17 antibody described herein may comprise the heavy chain constant region having the sequence of SEQ ID NO: 31. In some embodiments, antibody Delta1 17 described herein may comprise the heavy chain constant region having the sequence of SEQ ID NO: 79. In some embodiments, the anti-Delta1-17 antibody described herein may comprise the heavy chain constant region having the sequence of SEQ ID NO: 80. In some embodiments, the anti-Delta1-17 antibody described herein may comprise the heavy chain constant region having the sequence of SEQ ID NO: 81. In some embodiments, the anti-Delta1-17 antibody described herein may comprise the heavy chain constant region having the sequence of SEQ ID NO: 82.

In some embodiments, the anti-Delta1 antibody has a light chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 83. In some embodiments, the anti-Delta1 antibody has a heavy chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 84. In some embodiments, the anti-Delta1 antibody has a heavy chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 85. In some embodiments, the anti-Delta1 antibody has a heavy chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 86. In some embodiments, the anti-Delta1 antibody has a heavy chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 87. In some embodiments, the anti-Delta1 antibody has a light chain sequence comprising SEQ ID NO: 83. In some embodiments, the anti-Delta1 antibody has a heavy chain sequence comprising SEQ ID NO: 84. In some embodiments, the anti-Delta1 antibody has a heavy chain sequence comprising SEQ ID NO: 85. In some embodiments, the anti-Delta1 antibody has a heavy chain sequence comprising SEQ ID NO: 86. In some embodiments, the anti-Delta1 antibody has a heavy chain sequence comprising SEQ ID NO: 87. In some embodiments, the isolated antibody has a light chain sequence consisting essentially of SEQ ID NO: 83 or consisting of SEQ ID NO: 83. In some embodiments, the isolated antibody has a heavy chain sequence consisting essentially of SEQ ID NO: 84 or consisting of SEQ ID NO: 84. In some embodiments, the isolated antibody has a heavy chain sequence consisting essentially of SEQ ID NO: 85 or consisting of SEQ ID NO: 85. In some embodiments, the isolated antibody has a heavy chain sequence consisting essentially of SEQ ID NO: 86 or consisting of SEQ ID NO: 86. In some embodiments, the isolated antibody has a heavy chain sequence consisting essentially of SEQ ID NO: 87 or consisting of SEQ ID NO: 87.

In some embodiments, the anti-Delta1 antibody has a light chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 83 and has a heavy chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 84. In some embodiments, the anti-Delta1 antibody has a light chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 83 and has a heavy chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 85. In some embodiments, the anti-Delta1 antibody has a light chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 83 and has a heavy chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 86. In some embodiments, the anti-Delta1 antibody has a light chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 83 and has a heavy chain sequence that is at least 80 or 85% (e.g., at least 80%, 81%, 82%, 83% 84% or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 87. In some embodiments, the isolated antibody has a light chain sequence comprising SEQ ID NO: 83 and a heavy chain sequence comprising SEQ ID NO: 84. In some embodiments, the isolated antibody has a light chain sequence comprising SEQ ID NO: 83 and a heavy chain sequence comprising SEQ ID NO: 85. In some embodiments, the isolated antibody has a light chain sequence comprising SEQ ID NO: 83 and a heavy chain sequence comprising SEQ ID NO: 86. In some embodiments, the isolated antibody has a light chain sequence comprising SEQ ID NO: 83 and a heavy chain sequence comprising SEQ ID NO: 87. In some embodiments, the isolated antibody has a light chain sequence consisting essentially of SEQ ID NO: 83 and a heavy chain sequence consisting essentially of SEQ ID NO: 84. In some embodiments, the isolated antibody has a light chain sequence consisting essentially of SEQ ID NO: 83 and a heavy chain sequence consisting essentially of SEQ ID NO: 85. In some embodiments, the isolated antibody has a light chain sequence consisting essentially of SEQ ID NO: 83 and a heavy chain sequence consisting essentially of SEQ ID NO: 86. In some embodiments, the isolated antibody has a light chain sequence consisting essentially of SEQ ID NO: 83 and a heavy chain sequence consisting essentially of SEQ ID NO: 87. In some embodiments, the isolated antibody has a light chain sequence consisting of SEQ ID NO: 83 and a heavy chain sequence consisting of SEQ ID NO: 84. In some embodiments, the isolated antibody has a light chain sequence consisting of SEQ ID NO: 83 and a heavy chain sequence consisting of SEQ ID NO: 85. In some embodiments, the isolated antibody has a light chain sequence consisting of SEQ ID NO: 83 and a heavy chain sequence consisting of SEQ ID NO: 86. In some embodiments, the isolated antibody has a light chain sequence consisting of SEQ ID NO: 83 and a heavy chain sequence consisting of SEQ ID NO: 87.

Alternatively, the heavy chain constant region of the antibodies described herein may comprise a single domain (e.g., C_(H)1, C_(H)2, or C_(H)3) or a combination of any of the single domains, of a constant region (e.g., SEQ ID NO: 31).

When needed, the anti-Delta1 antibody as described herein may comprise a modified constant region. For example, it may comprise a modified constant region that is immunologically inert, e.g., does not trigger complement mediated lysis, or does not stimulate antibody-dependent cell mediated cytotoxicity (ADCC). ADCC activity can be assessed using methods disclosed in U.S. Pat. No. 5,500,362. In other embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8. In other examples, the antibody described herein may contain a modified constant region having an enhanced ADCC activity.

Any of the anti-Delta1 antibodies described herein may comprise a light chain that further comprises a light chain constant region, which can be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain.

Antibody heavy and light chain constant regions are well known in the art, e.g., those provided in the IMGT database (www.imgt.org) or at www.vbase2.org/vbstat.php., both of which are incorporated by reference herein.

Preparation of Anti-Delta1 Antibodies

Antibodies capable of binding the delta1 chain of a γδ TCR as described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

In some embodiments, antibodies specific to a target antigen (e.g., a delta1 chain of a suitable species such as human or a fragment thereof) can be made by the conventional hybridoma technology. The full-length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal for generating antibodies binding to that antigen. The route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for production of mouse, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of immunogen, including as described herein.

Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, may be used in the hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After the fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parent cells. Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV immortalized B cells may be used to produce the anti-Delta1 monoclonal antibodies described herein. The hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass all derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies capable of interfering with Delta1 (γδ T cell) activity. Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body fluids by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired. Undesired activity if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen. Immunization of a host animal with a target antigen or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R₁N═C═NR, where R and R₁ are different alkyl groups, can yield a population of antibodies (e.g., monoclonal antibodies).

If desired, an antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma) may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use. In an alternative, the polynucleotide sequence may be used for genetic manipulation to “humanize” the antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody. For example, the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically manipulate the antibody sequence to obtain greater affinity to the target antigen and greater efficacy in inhibiting the activity of a target γδ1 TCR (thus the activity of the target γδ T cells). It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

In other embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are Xenomouse® from Amgen, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, N.J.). In another alternative, antibodies may be made recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, the phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

Alternatively, antibodies capable of binding to the target antigens as described herein may be isolated from a suitable antibody library via routine practice. Antibody libraries, which contain a plurality of antibody components, can be used to identify antibodies that bind to a specific target antigen (e.g., an epitope of a delta1 chain in this case) following routine selection processes as known in the art. In the selection process, an antibody library can be probed with the target antigen or a fragment thereof and members of the library that are capable of binding to the target antigen can be isolated, typically by retention on a support. Such screening process may be performed by multiple rounds (e.g., including both positive and negative selections) to enrich the pool of antibodies capable of binding to the target antigen. Individual clones of the enriched pool can then be isolated and further characterized to identify those having desired binding activity and biological activity. Sequences of the heavy chain and light chain variable domains can also be determined via conventional methodology.

There are a number of routine methods known in the art to identify and isolate antibodies capable of binding to the target antigens described herein, including phage display, yeast display, ribosomal display, or mammalian display technology.

As an example, phage displays typically use a covalent linkage to bind the protein (e.g., antibody) component to a bacteriophage coat protein. The linkage results from translation of a nucleic acid encoding the antibody component fused to the coat protein. The linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon. Phage display is described, for example, in U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8 and Hoet et al. (2005) Nat Biotechnol. 23(3)344-8. Bacteriophage displaying the protein component can be grown and harvested using standard phage preparatory methods, e.g. PEG precipitation from growth media. After selection of individual display phages, the nucleic acid encoding the selected protein components can be isolated from cells infected with the selected phages or from the phage themselves, after amplification. Individual colonies or plaques can be selected, and then the nucleic acid may be isolated and sequenced.

Other display formats include cell-based display (see, e.g., WO 03/029456), protein-nucleic acid fusions (see, e.g., U.S. Pat. No. 6,207,446), ribosome display (See, e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; and Schaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35), and E. coli periplasmic display (J Immunol Methods. 2005 Nov. 22; PMID: 16337958), and yeast display (Feldhaus et al., Nat Biotechnol. 2003; 21:163-70).

After display library members are isolated for binding to the target antigen, each isolated library member can be also tested for its ability to bind to a non-target molecule to evaluate its binding specificity. Examples of non-target molecules include streptavidin on magnetic beads, blocking agents such as bovine serum albumin, non-fat bovine milk, soy protein, any capturing or target immobilizing monoclonal antibody, or non-transfected cells which do not express the target. A high-throughput ELISA screen can be used to obtain the data, for example. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target as well as for cross-species reactivity to related targets or subunits of the target antigen. The non-target and target binding data are compared (e.g., using a computer and software) to identify library members that specifically bind to the target.

After selecting candidate library members that bind to a target, each candidate library member can be further analyzed, e.g., to further characterize its binding properties for the target, e.g., Delta1 chain (referred to herein as Delta1). Each candidate library member can be subjected to one or more secondary screening assays. The assay can be for a binding property, a catalytic property, an inhibitory property, a physiological property (e.g., cytotoxicity, renal clearance, immunogenicity), a structural property (e.g., stability, conformation, oligomerization state) or another functional property. The same assay can be used repeatedly, but with varying conditions, e.g., to determine pH, ionic, or thermal sensitivities.

As appropriate, the assays can use a display library member directly, a recombinant polypeptide produced from the nucleic acid encoding the selected polypeptide, or a synthetic peptide synthesized based on the sequence of the selected polypeptide. In the case of selected Fabs, the Fabs can be evaluated or can be modified and produced as intact IgG proteins. Exemplary assays for binding properties are described below.

Binding proteins can also be evaluated using an ELISA assay. For example, each protein is contacted to a microtitre plate whose bottom surface has been coated with the target, e.g., a limiting amount of the target. The plate is washed with buffer to remove non-specifically bound polypeptides. Then the amount of the binding protein bound to the target on the plate is determined by probing the plate with an antibody that can recognize the binding protein, e.g., a tag or constant portion of the binding protein. The antibody is linked to a detection system (e.g., an enzyme such as alkaline phosphatase or horse radish peroxidase (HRP) which produces a colorimetric product when appropriate substrates are provided).

Alternatively, the ability of a binding protein described herein to bind a target antigen can be analyzed using a homogenous assay, i.e., after all components of the assay are added, additional fluid manipulations are not required. For example, fluorescence resonance energy transfer (FRET) can be used as a homogenous assay (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first molecule (e.g., the molecule identified in the fraction) is selected such that its emitted fluorescent energy can be absorbed by a fluorescent label on a second molecule (e.g., the target) if the second molecule is in proximity to the first molecule. The fluorescent label on the second molecule fluoresces when it absorbs to the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. A binding event that is configured for monitoring by FRET can be conveniently measured through standard fluorometric detection means, e.g., using a fluorimeter. By titrating the amount of the first or second binding molecule, a binding curve can be generated to estimate the equilibrium binding constant.

Surface plasmon resonance (SPR) can be used to analyze the interaction of a binding protein and a target antigen. SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of SPR). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Pat. No. 5,641,640; Raether, 1988, Surface Plasmons Springer Verlag; Sjolander and Urbaniczky, 1991, Anal. Chem. 63:2338-2345; Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705 and on-line resources provide by BIAcore International AB (Uppsala, Sweden).

Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (K_(D)), and kinetic parameters, including K_(on) and K_(off), for the binding of a binding protein to a target. Such data can be used to compare different biomolecules. For example, selected proteins from an expression library can be compared to identify proteins that have high affinity for the target or that have a slow K_(off). This information can also be used to develop structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of matured versions of a parent protein can be compared to the parameters of the parent protein. Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity and slow K_(off). This information can be combined with structural modeling (e.g., using homology modeling, energy minimization, or structure determination by x-ray crystallography or NMR). As a result, an understanding of the physical interaction between the protein and its target can be formulated and used to guide other design processes.

As a further example, cellular assays may be used. Binding proteins can be screened for ability to bind to cells which transiently or stably express and display the target of interest on the cell surface. For example, Delta1 binding proteins can be fluorescently labeled and binding to Delta1 in the presence or absence of antagonistic antibody can be detected by a change in fluorescence intensity using flow cytometry e.g., a FACS machine.

Antigen-binding fragments of an intact antibody (full-length antibody) can be prepared via routine methods. For example, F(ab′)2 fragments can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab′)2 fragments.

Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bi-specific antibodies, can be produced via, e.g., conventional recombinant technology. In one example, DNA encoding a monoclonal antibodies specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). Once isolated, the DNA may be placed into one or more expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. See, e.g., PCT Publication No. WO 87/04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, genetically engineered antibodies, such as “chimeric” or “hybrid” antibodies; can be prepared that have the binding specificity of a target antigen.

Techniques developed for the production of “chimeric antibodies” are well known in the art. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452.

Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, variable regions of V_(H) and V_(L) of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human V_(H) and V_(L) chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent V_(H) and V_(L) sequences as search queries. Human V_(H) and V_(L) acceptor genes are then selected.

The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions (see above description) can be used to substitute for the corresponding residues in the human acceptor genes.

A single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage or yeast scFv library and scFv clones specific to Delta1 can be identified from the library following routine procedures. Positive clones can be subjected to further screening to identify those that inhibit γδ T cell activity.

In some examples, any of the anti-Delta1 antibodies described herein can be a binding moiety of a bi-specific or tri-specific antibody. In other examples, any of the anti-Delta1 antibodies can be used for constructing a chimeric antigen receptor (CAR), which can be expressed on immune cells such as T cells. Any bi-specific antibodies or CAR-T cells comprising the anti-Delta1 antibodies are also within the scope of the present disclosure.

Antibodies obtained following a method known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping.” There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an additional example, epitope mapping can be used to determine the sequence, to which an antibody binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence). Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an antibody. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to the gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined. The gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, domain swapping experiments can be performed using a mutant of a target antigen in which various fragments of the Delta1 polypeptide have been replaced (swapped) with sequences from a closely related, but antigenically distinct protein (such as another member of the β-galactoside-binding soluble lectin family). By assessing binding of the antibody to the mutant Delta1, the importance of the particular antigen fragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.

In some examples, an anti-Delta1 antibody is prepared by recombinant technology as exemplified below.

Nucleic acids encoding the heavy and light chain of an anti-Delta1 antibody as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct prompter. Alternatively, the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter. When necessary, an internal ribosomal entry site (IRES) can be inserted between the heavy chain and light chain encoding sequences.

In some examples, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.

Generally, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors/promoter would depend on the type of host cells for use in producing the antibodies.

A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter.

Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. The tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16):1392-1399 (2003)). One particular advantage of this tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.

Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

Examples of polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.

One or more vectors (e.g., expression vectors) comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies. The host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification. If necessary, polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.

In some embodiments, methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an anti-Delta1 antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., a dhfr− CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium. When necessary, the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.

In one example, two recombinant expression vectors are provided, one encoding the heavy chain of the anti-Delta1 antibody and the other encoding the light chain of the anti-Delta1 antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr− CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody. When the two expression vectors are introduced into the same host cells, the antibody produced therein can be recovered from the host cells or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.

Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.

Any of the nucleic acids encoding the heavy chain, the light chain, or both of an anti-Delta1 antibody as described herein, vectors (e.g., expression vectors) containing such; and host cells comprising the vectors are within the scope of the present disclosure.

Anti-Delta1 antibodies thus prepared can be can be characterized using methods known in the art, whereby reduction, amelioration, or neutralization of γδ T cell biological activity is detected and/or measured. For example, an ELISA-type assay may be suitable for qualitative or quantitative measurement of γδ T cell inhibition of αβ T cell activation or lack thereof.

The bioactivity of an anti-Delta1 antibody can verified by incubating a candidate antibody with stimulated conventional (αβ) T cells and isolated γδ T cells, and monitoring any one or more of the following characteristics: (a) binding between the candidate antibody and γδ T cells and a subsequent increase in the level of T cell activation markers, indicating that the antibody blocks the inhibitory function of the γδ T cells; (b) preventing, ameliorating, or treating any aspect of a solid tumor; (c) blocking or decreasing γδ T cell activation; and (d) inhibiting (reducing) synthesis, production or release of γδ T cells.

Pharmaceutical Compositions and Uses Thereof

The present disclosure provides pharmaceutical compositions comprising the anti-Delta1 antibody described herein and uses of such for inhibiting signaling mediated by γδ1 T cells or activity mediated by γδ1 T cells, and/or eliminating Delta1-positive cells. Such antibodies can be used for treating diseases associated with activated γδ1 T cells, or for determining presence/level of γδ T cells in a biological sample.

Pharmaceutical Compositions

The antibodies, as well as the encoding nucleic acids or nucleic acid sets, vectors comprising such, or host cells comprising the vectors, as described herein can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington:

The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS' or polyethylene glycol (PEG).

In some examples, the pharmaceutical composition described herein comprises liposomes containing the antibodies (or the encoding nucleic acids) which can be prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

The antibodies, or the encoding nucleic acid(s), may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethyl cellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein can be formulated in sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT′ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%.

The emulsion compositions can be those prepared by mixing an antibody with Intralipid™ or the components thereof (soybean oil, egg phospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

Therapeutic Applications

The present disclosure provides pharmaceutical compositions comprising at least one anti-Delta1 antibody described herein or antigen binding fragment thereof and uses of such for inhibiting and/or reducing activity and/or reducing signaling mediated by a γδ1TCR expressing T cells and/or eliminating or reducing number(s) of γδ1 T cells. In some embodiments, the antibodies are useful for treating diseases associated with γδ1 T cells. Any of the anti-Delta1 antibodies described herein can be used in the methods described herein. In some embodiments, the anti-Delta1 antibody is selected from Delta1-1, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or combinations thereof. In some embodiments, the anti-Delta1 antibody is selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17 or combinations thereof. Non-limiting examples of such antibodies include for example Delta1-17. In some aspects, the invention provides methods of treating cancer. In some embodiments, the present disclosure methods for reducing, ameliorating, or eliminating one or more symptom(s), and/or extending survival (disease-free, progression-free, and/or overall survival), associated with cancer.

In some embodiments, the disclosure provides a method for treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the disclosure provides a method for treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is selected from Delta1-1, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or combinations thereof. A non-limiting example of such antibodies includes Delta1-17.

The present disclosure provides methods of inhibiting γδ1 TCR-mediated cell signaling in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an anti-Delta1 antibody described herein, including, but not limited to Delta1-17.

To practice the method disclosed herein, an effective amount of the pharmaceutical composition described herein is administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, the anti-Delta1 antibodies as described herein are aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

In some embodiments, the subject to be treated by the methods described herein is a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a target disease/disorder, such as a solid tumor.

In some embodiments, the cancer is selected from adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma tumors, osteosarcoma, malignant fibrous histiocytoma), brain cancer (e.g., astrocytomas, brain stem glioma, craniopharyngioma, ependymoma), bronchial tumors, cholangiocarcinoma, cholangiosarcoma, central nervous system tumors, breast cancer, Castleman disease, cervical cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, genitourinary cancers, gestational trophoblastic disease, heart cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, hypopharyngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia), liver cancer, lung cancer (for example, non-small cell lung cancer, NSCLC, and small cell lung cancer, SCLC), lymphoma (e.g., AIDS-related lymphoma, Burkitt lymphoma, cutaneous T cell lymphoma, Hogkin lymphoma, Non-Hogkin lymphoma, primary central nervous system lymphoma), malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity cancer, paranasal sinus cancer, pancreatic duct adenocarcinoma (PDA) nasopharyngeal cancer, neuroblastoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma, liposarcoma, lipomyosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, rhabdoid tumor, salivary gland cancer, sarcoma, skin cancer (e.g., basal cell carcinoma, melanoma), squamous cell head and neck cancer, small intestine cancer, stomach cancer, teratoid tumor, testicular cancer, throat cancer, thymus cancer, thyroid cancer, unusual childhood cancers, upper and lower gastrointestinal malignancies (including, but not limited to, esophageal, gastric, and hepatobiliary cancer), urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, cancers of the unknown primary, Waldenstrom macroglobulinemia, and Wilms tumor. In some embodiments, the cancer is selected from hematological malignancies include acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas, multiple myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndromes and the myeloproliferative neoplasms, such as essential thrombocythemia, polycythemia vera and myelofibrosis. In some embodiments, the symptom(s) associated thereof include, but are not limited to, anemia, loss of appetite, irritation of bladder lining, bleeding and bruising (thrombocytopenia), changes in taste or smell, constipation, diarrhea, dry mouth, dysphagia, edema, fatigue, hair loss (alopecia), infection, infertility, lymphedema, mouth sores, nausea, pain, peripheral neuropathy, tooth decay, urinary tract infections, and/or problems with memory and concentration. The method may comprise preparing a pharmaceutical composition with an anti-Delta1 antibody described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. In certain embodiments, administering the pharmaceutical composition, e.g., one or more of the anti-Delta1 antibodies described herein, including but not limited to antibody Delta1-17, to the subject reduces cell proliferation, tumor growth, and/or tumor volume in a subject, or reduces the number of metastatic lesions over time. In some embodiments, administering the composition results in complete response, partial response, or stable disease.

Examples of solid tumor cancers include pancreatic duct adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer (for example, non-small cell lung cancer, NSCLC, and small cell lung cancer, SCLC), glioblastoma, upper and lower gastrointestinal malignancies (including, but not limited to, esophageal, gastric, colorectal, pancreatic, bile duct (cholangiocarcinoma), and hepatobiliary cancer), squamous cell head and neck cancer, genitourinary cancers, endometrial cancer, renal cancer, bladder cancer, prostate cancer, ovarian cancer, neuroendocrine cancer (carcinoid and pancreatic neuroendocrine tumors), adrenocortical cancer, and sarcomas. Hematological malignancies include acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas, multiple myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndromes and the myeloproliferative neoplasms, such as essential thrombocythemia, polycythemia vera and myelofibrosis. A subject having a solid tumor or a hematological malignancy can be identified by routine medical examination, e.g., laboratory tests, organ functional tests relevant imaging modalities. In some embodiments, the subject to be treated by the method described herein may be a human cancer patient who has undergone or is subjecting to an anti-cancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, cell-based therapy, surgery or any combination thereof.

Increased numbers of γδ T cells have been found in a number of cancers, including, but not limited to, glioma, melanoma, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, neuroendocrine tumors (e.g., carcinoid tumors), breast cancer, lung cancer, ovarian cancer, renal cancer, bladder cancer, and prostate cancer. In some cases, as explained below, the proportion or number of γδ1 T cells is increased in cancers relative to a non-cancerous control, and/or the proportion or number of γδ2 T cells is decreased in the cancer relative to a non-cancerous control. Without wishing to be bound by theory, blocking or targeting delta1 chain TCRs and thereby reducing an immunosuppressive function of γδ T cells expressing the delta1 TCRs (Vδ1 cells), e.g., by using or administering an anti-Delta1 antibody, may present an effective novel therapeutic approach for the treatment of certain cancers, e.g., cancers with high levels of γδ T cells. Accordingly, any of the anti-delta-1 antibodies disclosed herein are suitable for inhibiting immune suppression by γδ T cells, and reactivating the effector T cell response. Accordingly, the anti-Delta1 antibodies described herein are suitable for the treatment of cancers, e.g., cancers associated with γδ T cells (e.g., cancers in which γδ T cells play a role in cancer development and progression). Accordingly, methods of treating cancer are provided herein, comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta-1 antibody being administered to the subject, is selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17 or functional variants thereof as disclosed herein. In one example, the antibody is Delta1-17.

Non-limiting exemplary cancers that are treated by the anti-delta1 antibodies disclosed herein are provided below.

Gliomas, tumors of the glial cells of the brain or the spine, are a deadly form of brain cancer, comprising about 80% of all malignant brain tumors and 50% of primary brain tumors (Goodenberger and Jenkins, Genetics of adult glioma. Cancer Genet. 2012 December; 205(12):613-21). The cancer is characterized by infiltrative growth of the tumor without boundary in the brain, leaving behind extensive necrosis, and frequently disrupting the blood-brain barrier. Glioblastoma (glioblastoma multiforme, GBM), is the most aggressive type of glioma. High-grade gliomas typically regrow despite complete surgical resection. Other treatments, such as radiotherapy and chemotherapy, have been used with very limited success.

It has been found that, while the ratio of total γδ T cells in peripheral blood in patients with glioma is not significantly different from that of healthy controls, the ratio of Vδ1 T cells is significantly higher while the ratio of γδ2 T cells is significantly lower in the peripheral blood of glioma patients, as compared to healthy controls (Liu et al., γδ T Cells in Peripheral Blood of Glioma Patients. Med Sci Monit. 2018; 24:1784-92). Without wishing to be bound by theory, blocking or targeting delta1, and thereby potentially reducing an immunosuppressive function of γδ1 cells, e.g., by administering an antibody that binds to delta1, may present a novel therapeutic approach for the treatment of glioma.

In some embodiments, the disclosure provides a method for treating glioma (e.g., glioblastoma) in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is glioma (e.g., glioblastoma).

Melanoma is the deadliest form of skin cancer and has been increasing in incidence for the past 30 years, especially in young adults. Accumulation of genetic disorders, most frequently mutations in B-Raf and N-Ras, in the melanocyte are a hallmark of melanoma (Rodriguez-Cerdeira et al., Advances in Immunotherapy for Melanoma: A Comprehensive Review; Mediators Inflamm. 2017; 2017: 3264217, and references therein). Subsequently, these alterations to result in the transformation of a dysplastic melanocyte into a melanoma cell, followed by invasion and metastasis.

Similar to glioma, frequencies of γδ1 cells are higher in melanoma patients compared to healthy controls (Wistuba-Hamprecht et al., Eur J Cancer. 2016 September; 64:116-26). This was found regardless of whether patients were treated with ipilimumab or not. In contrast, levels of Vδ2 cells were lower in melanoma patients as compared to healthy controls and ipilimumab reduced γδ2 cell levels in patients with worse outcomes. In this study, high frequencies of γδ2 cells and low frequencies of γδ1 cells were associated with favorable overall survival (OS) of melanoma patients. Without wishing to be bound by theory, blocking or targeting delta1 and potentially reducing an immunosuppressive function of γδ1 cells, e.g., by administering an antibody that binds to delta1, may present a novel therapeutic approach in melanoma, which can lead to improved overall survival in patients, including but not limited to those treated with ipilimimab.

In some embodiments, the disclosure provides a method for treating melanoma in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is melanoma.

Sarcomas are tumors of mesenchymal (connective) tissue, and include malignant tumors of bone (e.g., osteosarcoma), cartilage (chondrosarcoma), fat (liposarcoma), muscle (e.g., leiomyosarcoma), vascular, and hematopoietic tissues. Sarcomas are typically treated with surgery, although chemotherapy and radiation may also be administered before and/or after surgery to improve outcome.

In some embodiments, the disclosure provides a method for treating a sarcoma in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is a sarcoma.

Gastrointestinal (GI) cancers include, but are not limited to, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, and liver cancer. GI cancers represent the largest number of cancers and most deaths from cancer than any other system of the body.

Esophageal cancer, which is the sixth most common cancer worldwide, is increasing in incidence. There are two main types of esophageal cancer: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), and while the cause of this cancer is unknown, certain risk factors, such as the use of tobacco or alcohol, as well as reflux, Barrett's esophagus, achalasia, Plummer-Vinson syndrome, or esophageal scarring, have been identified (American Cancer Society, Esophageal Cancer, Jun. 14, 2017). Current treatments usually include surgery, as well as chemotherapy, radiotherapy, and/or stenting (Short et al., Esophageal Cancer. Am Fam Physician. 2017 Jan. 1; 95(1):22-28).

It has been found that adhesion molecules recruit γδ1 T cells from the peripheral blood to the tumor tissue in esophageal patients, as γδ1 T cells are found to be sequestered in tumor tissue of patients having esophageal cancer (Thomas et al., Role of adhesion molecules in recruitment of γδ1 T cells from the peripheral blood to the tumor tissue of esophageal cancer patients. Cancer Immunol Immunother. 2001 June; 50(4):218-25). Given the increased levels of γδ1 T cells, blocking or targeting delta1 e.g., by administering an antibody that binds to delta1, may present a novel therapeutic approach in esophageal cancer, which can lead to improved overall survival in patients.

In some embodiments, the disclosure provides a method for treating esophageal cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is esophageal cancer.

Gastric cancer, which develops in the lining of the stomach, used to be the leading cause of cancer deaths until the 1980s, and is currently the third most common cause of cancer-related death in the world (World Health Organization, Fact Sheets—Cancer, Sep. 12, 2018). There are a number of factors that lead to gastric cancer, including Helicobacter pylori infection, smoking, diet, and genetics. One genetic risk factor for gastric cancer is a genetic defect of the CDH1 gene. Generally, treatment of gastric cancer includes surgery, chemotherapy, and radiation therapy, biologics, immunotherapy for a limited set of gastric cancers, although cures are rare. Treatment with a human epidermal growth factor receptor 2 (HER2) inhibitor, trastuzumab, has been found to increase overall survival in patients with inoperable locally advanced or metastatic gastric carcinoma over-expressing the HER2/neu gene (Orditura et al., Treatment of gastric cancer. World J Gastroenterol. 2014 Feb. 21; 20(7): 1635-1649). However, further anti-treatment strategies are needed to improve outcomes for gastric cancer patients.

It has been found that γδ T cells promote gastric cancer development. In particular, γδ T cells are a major source of IL-17 in the tumor microenvironment, and IL-17 supports angiogenesis in gastric cancer, promoting cancer growth (Wu et al., IL-17 promotes tumor angiogenesis through Stat3 pathway mediated upregulation of VEGF in gastric cancer. Tumour Biol. 2016 April; 37(4):5493-501). Therefore, without wishing to be bound by theory, γδT cells are key producers of immune suppressive cytokines in gastric cancer and a present a novel target for immunotherapy.

In some embodiments, the disclosure provides a method for treating gastric cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is gastric cancer.

Colorectal cancer (CRC), also known as bowel cancer, colon cancer, or rectal cancer, is any cancer affecting the colon and the rectum. CRC is known to be driven by genetic alterations of tumor cells and is also influenced by tumor-host interactions. Recent reports have demonstrated a direct correlation between the densities of certain T lymphocyte subpopulations and a favorable clinical outcome in CRC, supporting a major role of T-cell-mediated immunity in repressing tumor progression of CRC. As for most cancers, current treatment for CRC includes surgery, chemotherapy, and radiation. In addition, drugs targeting specific mutations (e.g., bevacizumab, cetuximab, panitumumab, ramucirumab, regorafenib, and ziv-aflibercept) may be administered. Immunotherapy antibodies, such as pembrolizumab and nivolumab may be administered as well. However, there exists a need for further anti-tumor treatments to improve patient outcome.

Frequencies of γδ1 cells are higher in rectal tumor tissues from rectal cancer patients, and were found to positively correlate with T stage (Rong et al., Analysis of tumor-infiltrating gamma delta T cells in rectal cancer. World J Gastroenterol. 2016 Apr. 7; 22(13): 3573-3580). In contrast, levels of γδ2 cells were lower in rectal cancer patients as compared to healthy controls, and negatively correlated with T stage. The tumor-infiltrating γδ1 T cells were found to have strong inhibitory effects, and it was concluded that the percentage imbalance in γδ1 and γδ2 T cells in rectal cancer patients may contribute to the development of rectal cancer. Without wishing to be bound by theory, blocking or targeting delta1 and potentially reducing an inhibitory function of γδ1 cells, e.g., by administering an antibody that binds to delta1, may present a novel therapeutic approach in CRC, which can lead to improved overall survival in patients.

In some embodiments, the disclosure provides a method for treating colorectal cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is colorectal cancer.

Pancreatic cancer, which includes pancreatic ductal adenocarcinoma (PDA), accounts for approximately 3% of all cancers and 7% of all cancer deaths in the US (American Cancer Socienty, 2019). In PDA, which accounts for about 85% of all pancreatic cancers, four genes have found to be mutated in the majority of cases: KRAS, CDKN2A, TP53, and SMAD4 (Wolfgang et al., CA Cancer J Clin. 2013 September; 63(5): 318-348). Treatment of pancreatic cancer typically consists of surgical resection and adjuvant therapy; currently the median overall survival for patients with resected pancreatic cancer is still approximately 20-22 months. Thus, there exists a need for additional anti-tumor strategies to further improve outcomes for pancreatic cancer patients.

In human pancreatic ductal adenocarcinoma (PDA), an activated γδT cell population constitutes up to 75% of tumor-infiltrating T cells (Daley et al., Cell. 2016 Sep. 8; 166(6):1485-1499.e15), and γδT cells produce high levels of tumor-promoting IL-17 in PDA (McAllister et al., Cancer Cell. 2014 May 12; 25(5):621-37). Deletion of intra-pancreatic γδT cells markedly protects against oncogenesis in vivo and results in an influx of immunogenic Th1 cells and CD8+ T cells to the tumor micro environment (TME). Without wishing to be bound by theory, pancreas-infiltrating γδT cells promote PDA progression by inducing adaptive immune suppression, and accordingly, γδT cells are key regulators of effector T cell activation in pancreatic cancer and a new target for cancer immunotherapy.

In some embodiments, the disclosure provides a method for treating pancreatic cancer (e.g., PDA) in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is pancreatic cancer (e.g., PDA).

Cholangiocarcinoma (CCA) is an epithelial cancer that forms in the bile ducts and is the most common biliary malignancy and the second most common hepatic malignancy after hepatocellular carcinoma. The overall incidence of cholangiocarcinoma has increased progressively worldwide over the past four decades. CCAs are classified into three subtypes based on their anatomic location, intrahepatic cholangiocarcinoma (iCCA), perihilar CCA (pCCA), and distal CCA (dCCA) (see, e.g., Loeuillard et al., Animal models of cholangiocarcinoma; Biochim Biophys Acta Mol Basis Dis. 2018 Apr. 5., and Rizvi et al., Cholangiocarcinoma—evolving concepts and therapeutic strategies; Nat Rev Clin Oncol. 2018 February; 15(2): 95-111). Currently, the disease is incurable and lethal, unless the tumor(s) can be fully resected in the early stages. Other most common treatments include adjuvant chemotherapy and radiation therapy.

In some embodiments, the disclosure provides a method for treating cholangiocarcinoma in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is cholangiocarcinoma.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. It is the sixth most frequent cancer and second leading cause of death from cancer. Hepatocellular carcinoma occurs most often in people with chronic liver diseases, such as cirrhosis caused by hepatitis B or hepatitis C infection. HCC is usually accompanied by cirrhotic liver with extensive lymphocyte infiltration due to chronic viral infection. Current treatments for liver cancer include partial surgical resection, liver transplantation, percutaneous ablation, localized (e.g., transarterial chemoembolization) and systemic chemotherapy, small molecule TKIs and immunotherapy. Additional anti-tumor treatments strategies are however needed to further improve liver cancer patients' outcomes.

It has been found that γδ T cells accumulate in liver tumors, as patients with hepatic malignancies have increased levels of γδ T cells as compared to healthy controls (Kenna et al., Distinct subpopulations of gamma delta T cells are present in normal and tumor-bearing human liver. Clin Immunol. 2004; 113:56-63 and Hammerich et al., World J Gastrointest Pathophysiol. 2014 May 15; 5(2): 107-113). In addition, different Vδ chains can lead γδ T cells to have protective or damaging effects. Vβ1 T cells were found to correlate with higher necroinflammatory scores in hepatitis C virus patients, which could be indicative of how such cells would perform in liver cancer (Rajoriya et al., Front Immunol. 2014; 5: 400). Without wishing to be bound by theory, blocking certain populations of γδ T cells, for example with an anti-Delta1 antibody, may present a novel therapeutic approach in liver cancer, potentially leading to increased overall survival of patients.

In some embodiments, the disclosure provides a method for treating liver cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is liver cancer.

Neuroendocrine tumors (NETs), which originate in neuroendocrine cells, most often occur in the intestine, but can also occur in the pancreas, lung, and other areas of the body. Carcinoid tumors, slow-growing neuroendocrine tumors occurring in the enterochromaffin cells in the gastrointestinal and bronchopulmonary systems, while rare, are the most type of common gastrointestinal neuroendocrine tumor. There are number of different types of carcinoid tumors, including bronchopulmonary, gastric, small intestinal, appendiceal, and colorectal carcinoid tumors. Typically, treatment includes surgical resection, hepatic chemoembolization (if applicable), and medical therapy (Pinchot et al., Carcinoid tumors. Oncologist. 2008 December; 13(12): 1255-1269). Patients have been found to respond to somatostatin analogues (Aparicio et al., Antitumor activity of somatostatin analogues in progressive metastatic neuroendocrine gastroenteropancreatic tumors. Gut. 1996; 38:430-438), in addition to some chemotherapeutic drugs (Maroun et al., J Curr Oncol. 2006 April; 13(2):67-76) and a limited number of small molecule inhibitors. However, additional anti-tumor strategies are needed to improve outcomes for NET and carcinoid tumor patients. In some embodiments, the disclosure provides a method for treating a neuroendocrine tumor (e.g., a carcinoid tumor) in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is a neuroendocrine tumor (e.g., a carcinoid tumor).

Breast cancer is the second leading cause of cancer deaths in women. It is caused by a genetic mutation in the DNA of breast cancer cells most frequent type of cancer in women. Depending on the severity of the cancer, treatments can include surgery, chemotherapy, hormone therapy (e.g., hormone-blocking therapy, selective estrogen receptor modulators, aromatase inhibitors), and/or radiation. However, additional anti-tumor strategies are needed to improve outcomes for NET and carcinoid tumor patients.

γδ1 cells were found to be the dominant tumor-infiltrating T cells in tumors cells from patients with breast cancer compared to normal controls (Peng et al., Tumor-infiltrating γδ T cells suppress T and dendritic cell function via mechanisms controlled by a unique toll-like receptor signaling pathway. Immunity. 2007 August; 27(2):334-48). In contrast, levels of Vδ2 cells were lower in breast cancer patients as compared to healthy controls. An additional study, which looked specifically at triple-negative breast cancer, found increased numbers of γδ T cells as compared to the levels in normal breast tissue (Hidalgo et al., Histological analysis of γδ T lymphocytes infiltrating human triple-negative breast carcinomas. Front Immunol. 2014; 5: 632). In fact, the Vδ1 T cell subtype has been found to promote tumor growth and spread through its immunosuppressive effects (Morrow et al., The role of gamma delta T lymphocytes in breast cancer: a review. Transl Res. 2019 January; 203:88-9δ). Without wishing to be bound by theory, blocking or targeting delta1 and potentially reducing the immunosuppressive effects of γδ1 T cells in breast cancer, e.g., by administering an antibody that binds to delta1, may present a novel therapeutic approach in breast cancer, which can lead to improved overall survival in patients.

In some embodiments, the disclosure provides a method for treating breast cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is breast cancer.

Lung cancer is the most common cause of cancer-related death in men and second most common cause of cancer-related death in women. Mutations in the Kras proto-oncogene have been implicated in about 30% of cancers, while mutations in c-MET, NKX2-1, LIB1, PIK3CA, and BRAF have also been implicated (Herbst et al., Lung cancer. N Eng J Med. 2008. 359 (13): 1367-80). Treatment for lung cancer varies depending on its severity, and can include surgery, radiotherapy, chemotherapy, targeted drug therapy (e.g., erlotinib, gefitinib, afatinib, denosumab), and bronchoscopy treatments. However, the prognosis for people with lung cancer is less than 20% for five years after diagnosis. Therefore, additional anti-tumor strategies to further improve patient outcomes are needed.

In a study of non-small cell lung cancer (NSCLC) patients, it was found that the γδ1 T cell populations were enriched relative to the γδ2 T cell populations (Bao et al., Characterization of γδ T cells in patients with non-small cell lung cancer. Oncol Lett. 2017 July; 14(1): 1133-1140). In another study, it was demonstrated that lung cancer cells overexpress tumor-infiltrating γδ T lymphocytes, and that the cells represent “a sizeable fraction” of the tumor-infiltrating cells in lung cancer (Ferrarini et al., Killing of laminin receptor-positive human lung cancers by tumor infiltrating lymphocytes bearing gammadelta(+) t-cell receptors. J Natl Cancer Inst. 1996 Apr. 3; 88(7):436-41). Without wishing to be bound by theory, blocking or targeting delta1 and potentially reducing the immunosuppressive effects of γδ1 T cells in breast cancer, e.g., by administering an antibody that binds to Delta-1, may present a novel therapeutic approach in lung cancer, which can lead to improved overall survival in patients.

In some embodiments, the disclosure provides a method for treating lung cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is lung cancer.

Genitourinary cancers include, for example, ovarian, endometrial, renal, bladder, and prostate cancers.

Ovarian cancer, which is the most common gynecologic cause of death in Europe and North America, has a diverse progression, making its treatment and management difficult. Generally, treatment is surgery followed by chemotherapy (e.g., platinum-based chemotherapy). Likewise, uterine cancer (e.g., endometrial cancer, uterine sarcoma), the most common gynecologic cancer in the United States, presents with a varied disease progression. Treatment generally comprises surgery, chemotherapy, hormonal therapy, and radiotherapy. Renal cancer (e.g., renal cell carcinoma, transitional cell carcinoma) accounts for approximately 2% of all cancers worldwide, and its highest prevalence is in North America. Treatment generally consists of surgery, biological therapies (e.g., everolimus, torisel, nexavar, sutent, axitinib), immunotherapy (e.g., interferon, interleukin-2), and sunitinib and pazopanib. Renal cancer is generally not responsive to chemotherapy or radiotherapy. Bladder cancer is one of the most common cancers, and is highly treatable if diagnosed early. Current treatments include surgery, chemotherapy, radiation therapy, and immunotherapy (e.g., Bacillus Calmette-Guerin (BCG), interferon alfa-2b, atezolizumab). Prostate cancer, which is the most common cancer and the second leading cause of cancer death among men in the United States, may be treated by surgery, radiation therapy, hormone therapy, chemotherapy, and/or immunotherapy. Therefore, there exists a need for further anti-tumor strategies in order to improve outcomes for genitourinary cancer patients.

In a study, γδ T cells were found to be present in the intratumoral T cells of primary advanced untreated ovarian serous carcinomas, while αβ T cells were not (Raspollini et al., Tumor-infiltrating γδT-lymphocytes are correlated with a brief disease-free interval in advanced ovarian serous carcinoma. Ann Oncol. 2005 April; 16(4):590-6). In a murine ovarian cancer model, γδ T cells were found to accumulate at the later stages of tumor progression (Rei et al. Murine CD27(−) Vgamma6(+) γδT cells producing IL-17A promote ovarian cancer growth via mobilization of protumor small peritoneal macrophages. Proc Natl Acad Sci USA 2014; 111: E3562-E3570). Increased levels of γδ1 T cells have been found in renal cell cancers as well as prostate cancer (Groh et al., Broad tumor-associated expression and recognition by tumor-derived γδ T cells of MICA and MICB. Proc Natl Acad Sci USA. 1999 Jun. 8; 96(12): 6879-6884). In contrast, the increase in Vδ2 T cells following administration of BCG has found to be beneficial in bladder cancer (Pauza et al., Gamma Delta T Cell Therapy for Cancer: It Is Good to be Local. Front Immunol. 2018; 9:1305).

In some embodiments, the disclosure provides a method for treating a genitourinary cancer (e.g., ovarian cancer, endometrial (uterine) cancer, renal cancer, bladder cancer, prostate cancer) in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is a genitourinary cancer (e.g., ovarian cancer, endometrial (uterine) cancer, renal cancer, bladder cancer, prostate cancer).

Lymphomas are cancers of the lymphocytes, and include chronic lymphocytic leukemia, cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma (Hodgkin's disease), non-Hodgkin's lymphoma, and Waldenstrom macroglobulinemia. Treatments for lymphoma include chemotherapy, radiation therapy, and immunotherapy. A rare type of lymphoma, γδ T cell lymphoma, is frequently fatal, although treatment with allogenic stem cell transplantation may be possible. However, there exists a need for additional anti-tumor therapies to further improve outcomes for patients with lymphoma.

In some embodiments, the disclosure provides a method for treating a lymphoma in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is a lymphoma.

Adrenocortical carcinoma is a rare but aggressive form of cancer. The cancer is treated with surgical resection, although most patients are not candidates for this treatment, and are instead treated with radiation and radiofrequency ablation. Chemotherapy (e.g., mitotane, cisplatin, doxorubicin, etoposide and mitotane, streptozotocin and mitotane) may also be administered; however, the overall survival rate remains low. As such, additional anti-tumor strategies are needed to further improve outcomes for patients with adrenocortical carcinoma.

In some embodiments, the disclosure provides a method for treating adrenocortical carcinoma in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Delta1 antibody described herein or antigen binding fragment thereof. In some embodiments, the anti-Delta1 antibody is one or more of the anti-Delta1 antibodies disclosed herein, e.g., selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein.

In some embodiments, the disclosure provides the use of an anti-Delta1 antibody as a medicament for the treatment of a cancer, wherein the anti-Delta1 antibody is selected from one or more of any of the antibodies described herein (e.g., Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or a functional variant thereof such as those described herein), and wherein the cancer is adrenocortical carcinoma.

A subject suspected of having any of such target disease/disorder may or may not show one or more symptoms of the disease/disorder. A subject at risk for the disease/disorder can be a subject having one or more of the risk factors for that disease/disorder.

As used herein, “an effective amount” refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is reduced γδ T cell activity and/or amount/expression or increased anti-tumor immune responses in the tumor microenvironment (e.g., increased αβ T cell activation and/or activity). Determination of whether an amount of the antibody achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a target disease/disorder. Alternatively, sustained continuous release formulations of an antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In one example, dosages for an anti-Delta1 antibody as described herein may be determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of the antagonist. To assess efficacy of the antagonist, an indicator of the disease/disorder can be followed.

Generally, for administration of any of the anti-Delta1 antibodies described herein, an initial candidate dosage can be about 2 mg/kg to about 10 mg/kg or 1 mg/kg to about 20 mg/kg. For the purpose of the present disclosure, a typical dosage might range from about any of 0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. In some embodiments, any of the anti-Delta1 antibodies described herein may be given to a subject in need of the treatment at one or more flat doses i.e., is not dependent on the subject's weight, body surface area, or other factors alike. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a target disease or disorder, or a symptom thereof. An exemplary dosing regimen comprises administering an initial dose of about 3 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the antibody, or followed by a maintenance dose of about 1 mg/kg every other week. However, other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. For example, dosing from one-four times a week is contemplated. In some embodiments, dosing ranging from about 3 μg/mg to about 2 mg/kg (such as about 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg, about 300 μg/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In other embodiments, a flat dose (giving a patient a defined amount of the antibody without taking into consideration body weight, body surface area and other factors alike) can be used for treatment of a target disease as described herein.

In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) can vary over time.

In some embodiments, for an adult patient of normal weight, doses ranging from about 0.3 to 5.00 mg/kg may be administered. In some examples, the dosage of the anti-Delta1 antibody described herein can be 10 mg/kg. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent, and other considerations well known in the art).

For the purpose of the present disclosure, the appropriate dosage of an antibody as described herein will depend on the specific antibody, antibodies, and/or non-antibody peptide (or compositions thereof) employed, the type and severity of the disease/disorder, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antagonist, and the discretion of the attending physician. Typically the clinician will administer an antibody, until a dosage is reached that achieves the desired result. In some embodiments, the desired result is an increase in anti-tumor immune response in the tumor microenvironment. Methods of determining whether a dosage resulted in the desired result would be evident to one of skill in the art. Administration of one or more antibodies can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of an antibody may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a target disease or disorder.

As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder.

Alleviating a target disease/disorder includes delaying the development or progression of the disease, or reducing disease severity or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require curative results. As used therein, “delaying” the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

“Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and/or recurrence.

In some embodiments, the anti-Delta1 antibodies described herein are administered to a subject in need of the treatment at an amount sufficient to inhibit the activity of γδ T cells. In some embodiments, the anti-Delta1 antibodies described herein are administered to a subject in need of the treatment at an amount sufficient to inhibit the activity of γδ T cells by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In other embodiments, the anti-Delta1 antibodies are administered in an amount effective in rescuing immune inhibition induced by γδ T cells. In other embodiments, the anti-Delta1 antibodies are administered in an amount effective in rescuing immune inhibition induced by γδ T cells by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater).

In some embodiments, the disclosure provides a method for reducing tumor volume, tumor size, and/or tumor burden, the method comprising providing or administering an anti-anti-Delta1 antibody described herein, e.g., in Table 1 and/or elsewhere herein, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the method reduces tumor volume, tumor size, and/or tumor burden by 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for reducing tumor volume, tumor size, and/or tumor burden. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need thereof at an amount sufficient to reduce tumor volume, tumor size, and/or tumor burden. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to reduce tumor volume, tumor size, and/or tumor burden by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for depleting target cells, e.g., γδ T cells, e.g. γδ1 T cells, in a tumor and/or in peripheral blood, the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1 and/or elsewhere herein, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the method depletes γδ T cells, e.g. γδ1 cells, by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for depleting target cells, e.g., γδ T cells, e.g. γδ1 T cells. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need thereof at an amount sufficient to deplete γδ T cells, e.g. γδ1 T cells, in a tumor and/or in peripheral blood. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to deplete γδ T cells, e.g. γδ1 T cells, in a tumor and/or in peripheral blood by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for modulating the ratio of γδ T cells, e.g. modulating, e.g., reducing, the ratio γδ1 T cells to γδ2 T cells, in a tumor, the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1 or elsewhere herein, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the method modulates, e.g., reduces, the ratio γδ1 T cells to γδ2 T cells in a tumor by 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for modulating the ratio of γδ T cells, e.g. modulating, e.g., reducing, the ratio γδ1 T cells to γδ2 T cells, in a tumor. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need thereof at an amount sufficient to modulate, e.g., reduce, the ratio γδ1 T cells to γδ2 T cells in a tumor. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to modulate, e.g., reduce, the ratio γδ1 T cells to γδ2 T cells in a tumor by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for modulating the ratio γδ1 T cells in PBMCs and/or the ratio γδ1 T cells in immune cells present in a tumor, e.g., isolated from a subject having a cancer, e.g. modulating, e.g., reducing, the ratio γδ1 T cells in PBMCs and/or the ratio γδ1 T cells in immune cells present in a tumor, the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-23. In some embodiments, the method modulates, e.g., reduces, the ratio γδ1 T cells in PBMCs and/or the ratio γδ1 T cells in immune cells present in a tumor by 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for modulating the ratio γδ1 T cells in PBMCs and/or the fraction of γδ1 T cells present in tumor localized immune cells. In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for reducing, the fraction γδ1 T cells present in PBMCs and/or the fraction of γδ1 T cells present in tumor localized immune cells. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need thereof at an amount sufficient to modulate, e.g., reduce, the fraction γδ1 T cells present in PBMCs and/or the fraction of γδ1 T cells present in tumor localized immune cells. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to modulate, e.g., reduce, the fraction γδ1 T cells present in PBMCs and/or the fraction of γδ1 T cells present in tumor localized immune cells by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for inducing cell cytotoxicity, such as ADCC, in target cells, wherein the target cells are immune suppressive immune cells, e.g., γδ T cells, e.g. γδ1 T cells, the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1 and/or elsewhere herein, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the method induces apoptosis in γδ T cells, e.g. γδ1 T cells by 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for inducing cell cytotoxicity, such as ADCC, in target cells, wherein the target cells are immune suppressive immune cells, e.g., γδ T cells, e.g. γδ1 T cells. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need thereof at an amount sufficient to promote ADCC of γδ T cells, e.g. γδ1 T cells, in a tumor. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote ADCC in γδ T cells, e.g. γδ1 T cells, in a tumor by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for inducing cell cytotoxicity such as complement-dependent cytotoxicity (CDC) against target cells expressing delta1, i.e., γδ T cells, e.g. γδ1 T cells, in a subject, the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the method induces cell cytotoxicity such as complement-dependent cytotoxicity (CDC) against γδ T cells, e.g. γδ1 T cells, 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for inducing cell cytotoxicity such as complement-dependent cytotoxicity (CDC) against target cells expressing delta1, i.e., γδ T cells, e.g. γδ1 T cells. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote CDC in γδ T cells, e.g. γδ1 T cells, in a tumor. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote CDC in γδ T cells, e.g. γδ1 T cells, in a tumor by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for inducing phagocytosis of target cells expressing delta1 (ADCP), the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the anti-Delta1 antibody increases phagocytosis of γδ T cells, e.g. γδ1 T cells, by 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote ADCP in γδ T cells, e.g. γδ1 T cells, in a tumor. In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for inducing phagocytosis of target cells expressing delta1 (ADCP). In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote ADCP in γδ T cells, e.g. γδ1 T cells, in a tumor by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for inducing T cell activation, in a tumor and/or in peripheral blood, e.g., by targeting tumor infiltrating γδ T cells, e.g. γδ1 T cells, the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the method promotes T cell activation in a tumor and/or in peripheral blood by 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, inducing T cell activation, in a tumor and/or in peripheral blood. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote T cell activation in a tumor and/or in peripheral blood. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote T cell activation in a tumor and/or in peripheral blood by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for promoting CD4+ cell activation in a tumor and/or in peripheral blood, the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the method promotes CD4+ cell activation in a tumor and/or in peripheral blood by 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for promoting CD4+ cell activation in a tumor and/or in peripheral blood. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote CD4+ T cell activation in a tumor and/or in peripheral blood. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote CD4+ T cell activation in a tumor and/or in peripheral blood by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for inducing CD44 expression in CD4+ cells, the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the method increases proinflammatory cytokine expression in a tumor and/or in peripheral blood, e.g., CD44, IFNγ, and/or TNF-α expression, in CD4+ cells by 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for inducing CD44 expression in CD4+ cells. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to induce proinflammatory cytokine expression in a tumor and/or in peripheral blood, e.g., CD44, IFNγ, and/or TNF-α expression, in CD4+ cells in a tumor. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to induce proinflammatory cytokine expression, e.g., CD44, IFNγ, and/or TNF-α expression, in CD4+ cells in a tumor and/or in peripheral blood by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for promoting CD8+ cell activation in a tumor and/or in peripheral blood, the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the method promotes CD8+ cell activation in a tumor and/or in peripheral blood by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for promoting CD8+ cell activation in a tumor and/or in peripheral blood. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote CD8+ T cell activation in a tumor and/or in peripheral blood. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to promote CD8+ T cell activation in a tumor and/or in peripheral blood by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

In some embodiments, the disclosure provides a method for inducing CD44 expression in CD8+ cells in a tumor and/or in peripheral blood, the method comprising providing or administering an anti-Delta1 antibody described herein, e.g., in Table 1, or antigen binding fragment thereof to a subject. In some embodiments, the anti-Delta1 antibody is Delta1-17. In some embodiments, the method increases proinflammatory cytokine expression, e.g., CD44, IFNγ, and/or TNF-α expression, in CD8+ cells in a tumor and/or in peripheral blood by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).

In some embodiments, the disclosure provides one or more anti-Delta1 antibodies described herein, including, but not limited to, Delta1-17, for inducing CD44 expression in CD8+ cells in a tumor and/or in peripheral blood. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to induce proinflammatory cytokine expression, e.g., CD44, IFNγ, and/or TNF-α expression, in CD8+ cells in a tumor and/or in peripheral blood. In some embodiments, the antibodies described herein, e.g., in Table 1, including, but not limited to, Delta1-17, are administered to a subject in need of the treatment at an amount sufficient to induce proinflammatory cytokine expression, e.g., CD44, IFNγ, and/or TNF-α expression, in CD8+ cells in a tumor and/or in peripheral blood by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein) in vivo (as compared to levels prior to treatment or in a control subject).

Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

Injectable compositions may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.

In one embodiment, an antibody is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the antibody or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat. No. 5,981,568.

Targeted delivery of therapeutic compositions containing an antisense polynucleotide, expression vector, or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods and Applications of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

Therapeutic compositions containing a polynucleotide (e.g., those encoding the antibodies described herein) are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA or more can also be used during a gene therapy protocol.

The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of the coding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

The particular dosage regimen, i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history.

In some embodiments, more than one antibody, or a combination of an antibody and another suitable therapeutic agent, may be administered to a subject in need of the treatment. The antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.

Treatment efficacy for a target disease/disorder can be assessed by methods well-known in the art.

Combination Therapy

Any of the anti-Delta1 antibodies described herein may be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, small molecule inhibitors, cell based therapies, anti-hormonal agents, immunotherapy, anti-metabolics, biological agents, including bio-similars, and/or any combination thereof, and so forth. Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy according to the present disclosure.

When co-administered with an additional therapeutic agent, suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

In some embodiments, the anti-Delta1 antibody, such as antibody Delta1-17, can be combined with other immunomodulatory treatments such as, e.g., inhibitors of a checkpoint molecule (e.g., PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, A2aR, TIGIT and VISTA), activators of a co-stimulatory receptor (e.g., DX40, GITR, CD137, CD40, CD27, and ICOS), and/or inhibitors of an innate immune cell target (e.g., KIR, NKG2A, CD96, TLR, IDO, and/or galectin-9). Without being bound by theory, it is thought that anti-Delta1 antibodies can reprogram immune responses against tumor cells via, e.g., inhibiting the activity of γδ T cells infiltrated into tumor microenvironment. Thus, combined use of an anti-Delta1 antibody and an immunomodulatory agent such as those described herein would be expected to significantly enhance anti-tumor efficacy.

In some embodiments, the treatment method further comprises administering to the subject an inhibitor of a checkpoint molecule, an activator of a co-stimulatory receptor, and/or an inhibitor of an innate immune cell target. In some embodiments, the treatment method further comprises administering to the subject an inhibitor of a checkpoint molecule. In some embodiments, the checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, A2aR, TIGIT and VISTA. In some embodiments, the treatment method further comprises administering to the subject an inhibitor of an activator of a co-stimulatory receptor, and/or an inhibitor of an innate immune cell target. In some embodiments, the co-stimulatory receptor is selected from the group consisting of OX40, GITR, CD137, CD40, CD27, and ICOS. In some embodiments, the treatment method further comprises administering to the subject an inhibitor of an innate immune cell target. In some embodiments, the innate immune cell target is selected from the group consisting of KIR, NKG2A, CD96, TLR, and IDO. In some embodiments, the anti-Delta1 antibody is selected from Delta1-1, Delta1-3, Delta1-6, Delta1-8, Delta1-9, Delta1-10, Delta1-12, Delta1-13, Delta1-14, Delta1-15, or Delta1-17, or combinations thereof. A non-limiting example of such antibodies include for example antibody Delta1-17. In any of these methods of treatment, the anti-Delta1 antibody is antibody Delta1-17.

In other embodiments, the anti-Delta1 antibody described herein can also be co-used with a chemotherapeutic agent, including alkylating agents, anthracyclines, cytoskeletal disruptors (Taxanes), epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I, inhibitors of topoisomerase II, kinase inhibitors, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids and derivatives thereof. Non-limiting examples include: (i) anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000)); (ii) a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof; and (iii) chemotherapeutic compounds such as, e.g., pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine), purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, nanoparticle albumin-bound paclitaxel (Abraxane), docetaxel), eribulin, vincristine, vinblastine, nocodazole, epothilones, and navelbine, epidipodophyllotoxins (etoposide and teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, irinotecan (e.g., irinotecan liposome injections such as Onivyde®), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signal transduction kinase inhibitors; nucleotide analog and thymidine phosphorylase inhibitors (e.g., trifluridine-tipiracil or Lonsurf®); mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors. The therapeutic agents described herein include any drug products containing the listed active ingredients and derivatives thereof, as well as any formulations (e.g., sustained release formulations) including those known in the art.

Alternatively or in addition, the anti-Delta1 antibody described herein can also be co-used with an anti-hypertension agent, which includes, but is not limited to, angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, Lisinopril, moexipril, perindopril, quinapril, ramipril, or trandolapril), angiotensin II receptor blockers (a.k.a. ARBs or Sartans, e.g., azilsartan, candesartan, eprosartan, irbesartan, telmisartan, valsartan, losartan, or olmesartan), beta blockers (e.g., β1-selective agents such as acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, metoprolol, nebivolol; β2-selective agents such as butaxamine or ICI-118,551; β3-selective agents such as SR 59230A; and non-selective agents such as propranolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol) calcium channel blockers (e.g., amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nisoldipine, or verapamil), direct renin inhibitors (e.g., aliskiren), or diuretics. See, also US20130287688, and Pinter et al., Sci Transl Med (2017) 9(410).

Additional useful agents can be found in, e.g., Physician's Desk Reference, 59.sup.th edition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20.sup.th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15.sup.th edition, (2001), McGraw Hill, N.Y.; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

It was reported that chemotherapy and/or immune therapy of solid tumors could enhance the level of immune modulators such as checkpoint molecules, resulting in suppressed immunity against tumor cells. Erisson et al., J. Translational Medicine (2016), 14:282; Grabosch et al., J. ImmunoTherapy of Cancer (2015), 3(suppl 2): P302; and Azad et al., EMBO J. (2016). Anti-Delta1 antibodies have been found to inhibit the activity of γδ T cells, leading to increased T cell activation, particularly in solid tumors. As such, the co-use of an anti-Delta1 antibody and a chemotherapeutic agent (e.g., gemcitabine) or immunotherapeutic agent (e.g., anti-PD-L1 antibody) would be expected to result in significantly enhanced therapeutic activity against solid tumors, such as PDA or CRC.

In some embodiments, the methods are provided, wherein the anti-Delta1 antibody described herein is capable of improving anti-tumor activity of the co-administered checkpoint inhibitors, and/or other anti-cancer agents. In some embodiments, the methods are provided, wherein the anti-Delta1 antibody described herein is capable of improving anti-tumor activity of the co-administered checkpoint inhibitors. In some embodiments, the methods are provided, wherein the anti-Delta1 antibody, such as any of the anti-Delta1 antibodies described herein in Table 1 and elsewhere, such as Delta1-17, is capable of improving anti-tumor activity, i.e., efficacy (reducing tumor proliferation, size, volume, weight, burden or load or reduction in number of metastatic lesions over time, improving stable disease, improved survival rate) of the co-administered checkpoint inhibitors (e.g., PD-1, PD-L1 and/or CTLA-4 or others listed herein or known in the art), e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to a checkpoint inhibitor therapy alone under the same conditions. In some embodiments, the anti-Delta1 antibody, such as any of the anti-Delta1 antibodies described herein in Table 1 and elsewhere, for example Delta1-17, is capable of improving anti-tumor activity, i.e., efficacy (reducing tumor proliferation, size, volume, weight, burden or load or reduction in number of metastatic lesions over time, improving stable disease, improved survival rate) of the co-administered checkpoint inhibitors (e.g., PD-land/or CTLA-4 e.g., PD-1, PD-L1 and/or CTLA-4 or others listed herein or known in the art), e.g., 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold, three-fold, four-fold, ten-fold more or more as compared to a checkpoint inhibitor therapy alone under the same conditions.

In some embodiments, the co-administered checkpoint inhibitors (e.g., PD-1, PD-L1 and/or CTLA-4 or others listed herein or known in the art) are capable of improving anti-tumor activity of the anti-Delta1 antibody described herein. In some embodiments, the co-administered checkpoint inhibitors (e.g., PD-1, PD-L1 and/or CTLA-4 or others listed herein or known in the art) are capable of improving anti-tumor activity of the anti-Delta1 antibody, such as any of the anti-Delta1 antibodies described herein in Table 1 and elsewhere, including but not limited to Delta1-17, anti-tumor activity, i.e., efficacy (reducing tumor proliferation, size, volume, weight, burden or load or reduction in number of metastatic lesions over time, improving stable disease, improved survival rate), e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to anti-delta1 therapy alone under the same conditions. In some embodiments, the co-administered checkpoint inhibitors (e.g., PD-1, PD-L1 and/or CTLA-4 or others listed herein or known in the art) are capable of improving anti-tumor activity, i.e., efficacy (reducing tumor proliferation, size, volume, weight, burden or load or reduction in number of metastatic lesions over time, improving stable disease, improved survival rate) of the anti-Delta1 antibody, such as any of the anti-Delta1 antibodies described herein in Table 1 and elsewhere, including but not limited to Delta1-17, e.g., 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold, three-fold, four-fold, ten-fold more or more as compared to an anti-delta1 therapy alone under the same conditions.

In some embodiments, the methods are provided, wherein the anti-Delta1 antibody described herein is capable of improving the ability of the immunotherapy to activate T cells. In some embodiments, the methods are provided, wherein the anti-Delta1 antibody, such as any of the anti-Delta1 antibodies described herein in Table 1 and elsewhere, including but not limited to, Delta1-17, is capable of improving the ability of the immunotherapy to activate T cells (e.g., as measured by cytokine markers described herein) (e.g., as described herein or known in the art), e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to a immunotherapy therapy alone under the same conditions. In some embodiments, the anti-Delta1 antibody is capable of improving the ability of the immunotherapy to activate T cells (e.g., as measured by cytokine markers described herein) (e.g., as described herein or known in the art), e.g., 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold, three-fold, four-fold, ten-fold more or more as compared to a immunotherapy therapy alone under the same conditions.

In some embodiments, the anti-Delta1 antibody is administered as a maintenance therapy.

In some embodiments, the methods are provided, wherein the co-administered immunotherapies are capable of improving the ability of the anti-Delta1 antibody described herein to activate T cells. In some embodiments, the methods are provided, wherein the co-administered immunotherapies (e.g., as described herein or known in the art) are capable of improving the ability of the anti-Delta1 antibody, such as any of the anti-Delta1 antibodies described herein in Table 1 and elsewhere, including but not limited to, Delta1-17, to activate T cells (e.g., as measured by cytokine markers described herein), e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to an anti-delta1 therapy alone under the same conditions. In some embodiments, the co-administered immunotherapies (e.g., as described herein or known in the art) are capable of improving the ability of the anti-Delta1 antibody to activate T cells (e.g., as measured by cytokine markers described herein), e.g., 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold, three-fold, four-fold, ten-fold more or more as compared to an anti-delta1 therapy alone under the same conditions.

In some embodiments, the methods are provided, wherein the anti-Delta1 antibody is capable of improving anti-tumor activity of the co-administered chemotherapeutic agents. In some embodiments, the methods are provided, wherein the anti-Delta1 antibody, such as any of the antibodies described herein in Table 1 and elsewhere, for example Delta1-17, is capable of improving anti-tumor activity, i.e., efficacy, (reducing tumor proliferation, size, volume, weight, burden or load or reduction in number of metastatic lesions over time, improving stable disease, improved survival rate) of the co-administered chemotherapeutic agents (e.g., as described herein or known in the art), e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to a chemotherapeutic agent therapy alone under the same conditions. In some embodiments, the anti-Delta1 antibody is capable of improving anti-tumor activity, i.e., efficacy (reducing tumor proliferation, size, volume, weight, burden or load or reduction in number of metastatic lesions over time, improving stable disease, improved survival rate) of the co-administered chemotherapeutic agents (e.g., as described herein or known in the art), e.g., 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold, three-fold, four-fold, ten-fold more or more as compared to a chemotherapeutic agent therapy alone under the same conditions.

In some embodiments, the methods are provided, wherein the co-administered chemotherapeutic agents are capable of improving anti-tumor activity of the anti-Delta1 antibody. In some embodiments, the methods are provided, wherein the co-administered chemotherapeutic agents (e.g., as described herein or known in the art) are capable of improving anti-tumor activity of the anti-Delta1 antibody, such as any of the antibodies described herein in Table 1 and elsewhere, for example Delta1-17, (reducing tumor proliferation, size, volume, weight, burden or load or reduction in number of metastatic lesions over time, improving stable disease, improved survival rate) by, e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to an anti-delta1 therapy alone under the same conditions. In some embodiments, the co-administered chemotherapeutic agents (e.g., as described herein or known in the art) are capable of improving anti-tumor activity, i.e., efficacy (reducing tumor proliferation, size, volume, weight, burden or load or reduction in number of metastatic lesions over time, improving stable disease, improved survival rate) of the anti-Delta1 antibody, e.g., 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold, three-fold, four-fold, ten-fold more or more as compared to an anti-delta1 therapy alone under the same conditions.

In some embodiments, methods are provided herein, wherein the anti-Delta1 antibody is capable of improving the ability of the chemotherapeutic agent to activate T cells. In some embodiments, methods are provided herein, wherein the anti-Delta1 antibody, such as any of the antibodies described herein in Table 1 and elsewhere, for example Delta1-17, is capable of improving the ability of the chemotherapeutic agent to activate T cells (e.g., as measured by cytokine markers described herein) (e.g., as described herein or known in the art), e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to a chemotherapeutic agent therapy alone under the same conditions. In some embodiments, the anti-Delta1 antibody is capable of improving the ability of the chemotherapeutic agent to activate T cells (e.g., as measured by cytokine markers described herein) (e.g., as described herein or known in the art e.g., 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold, three-fold, four-fold, ten-fold more or more as compared to a chemotherapeutic agent therapy alone under the same conditions.

In some embodiments, methods are provided herein, wherein the co-administered chemotherapeutic agents are capable of improving the ability of the anti-Delta1 antibody to activate T cells. In some embodiments, methods are provided herein, wherein the co-administered chemotherapeutic agents (e.g., as described herein or known in the art) are capable of improving the ability of the anti-Delta1 antibody, such as any of the antibodies described herein in Table 1 and elsewhere, for example Delta1-17, to activate T cells (e.g., as measured by cytokine markers described herein), e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to an anti-delta1 therapy alone under the same conditions. In some embodiments, the co-administered chemotherapeutic agents (e.g., as described herein or known in the art) are capable of improving the ability of the anti-Delta1 antibody to activate T cells (e.g., as measured by cytokine markers described herein), e.g., 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold, three-fold, four-fold, ten-fold more or more as compared to an anti-delta1 therapy alone under the same conditions.

In any of these combination therapy methods the cancer to be treated is selected from Examples of solid tumor cancers include pancreatic duct adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer (for example, non-small cell lung cancer, NSCLC, and small cell lung cancer, SCLC), glioblastoma, upper and lower gastrointestinal malignancies (including, but not limited to, esophageal, gastric, colorectal, pancreatic, bile duct (cholangiocarcinoma), and hepatobiliary cancer), squamous cell head and neck cancer, genitourinary cancers, endometrial cancer, renal cancer, bladder cancer, prostate cancer, ovarian cancer, neuroendocrine cancer (carcinoid and pancreatic neuroendocrine tumors), adrenocortical cancer, and sarcomas. Hematological malignancies include acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas, multiple myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndromes and the myeloproliferative neoplasms, such as essential thrombocythemia, polycythemia vera and myelofibrosis, and others described herein.

Other Applications

Any of the anti-Delta1 antibodies described herein may also be used for detecting presence of γδ1 T cells and/or measuring the level of γδ1 T cells in a sample, such as a biological sample, using a conventional immunoassay (e.g., Western blotting, FACS, immunohistochemistry, immunocytochemistry, etc.). Briefly, the antibody can be incubated with a sample for a suitable period of time under suitable conditions allowing for binding of the antibody to the target γδ1 if present in the sample. Such a binding can then be detected using a conventional method, for example, using a labeled second antibody that binds the anti-Delta1 antibody. Performing such an immunoassay for detecting the target γδ1 TCR or T cells expressing such a TCR is well within the knowledge of those skilled in the art.

Kits for Use in Treatment of Diseases Associated with γδ T Cell Activation

The present disclosure also provides kits for use in treating or alleviating a disease associated with γδ T cell activation. Examples include solid tumors such as PDA and others described herein. Such kits can include one or more containers comprising an anti-Delta1 antibody, e.g., any of those described herein, and optionally a second therapeutic agent to be co-used with the anti-Delta1 antibody, which is also described herein.

In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of the anti-Delta1 antibody, and optionally the second therapeutic agent, to treat, delay the onset, or alleviate a target disease as those described herein. The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease, e.g., applying the diagnostic method as described herein. In still other embodiments, the instructions comprise a description of administering an antibody to an individual at risk of the target disease.

The instructions relating to the use of an anti-Delta1 antibody generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating the disease associated with γδ T cell activation. Instructions may be provided for practicing any of the methods described herein.

The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-Delta1 antibody as those described herein.

Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above.

General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES Example 1: Generation of Anti-Delta1 Antibodies

Antigen Production

Expression vectors for the gamma and delta chains of human and cynomolgus T cell receptors (TCR) fused to the Fc portion of mouse immunoglobulin G (IgG) were constructed using standard recombinant DNA methods. To make purification and immobilization more efficient, the expression vectors for the delta chains were further modified to include an AviTag™ and a His6 tag attached to the C-terminus of the Fc portion. Expression vectors for six different delta1 clones (human Delta1A (SEQ ID NO: 26), human Delta1B (SEQ ID NO: 27), human Delta1C (SEQ ID NO: 28), cynomolgus monkey Delta1A (SEQ ID NO: 32), cynomolgus monkey Delta1B (SEQ ID NO: 33), and cynomolgus monkey Delta1C (SEQ ID NO: 34)), four delta2 clones (human Delta2A (SEQ ID NO: 29), human Delta2B (SEQ ID NO: 35), human Delta2C (SEQ ID NO: 36), and cynomolgus monkey Delta2 (SEQ ID NO: 37)), and six gamma9 clones (human Gamma9 (SEQ ID NO: 30), human Gamma3 (SEQ ID NO: 38), human Gamma4 (SEQ ID NO: 39), human Gamma5 (SEQ ID NO: 40), human Gamma8 (SEQ ID NO: 41), cynomolgus monkey gamma (SEQ ID NO: 42)) were generated. The amino acid sequences of the extracellular region of the Delta1 chains are provided above and the amino acid sequences of the Delta2 and Gamma chains are given below. Mammalian cells were transfected with the vector for a delta chain clone (one of the 10 vectors described above) and a vector for the gamma chain (one of six gamma chains) to produce gamma/delta heterodimers. The resulting gamma/delta TCR-Fc fusion protein was extracted from the culture supernatant and purified using Ni Sepharose® chromatography followed by gel filtration to apparent homogeneity. The fusion protein was then biotinylated in vitro using recombinant BirA.

Human TCR Delta2A: (SEQ ID NO: 29) AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIY REKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDTLGMG GEYTDKLIFGKGTRVTVEPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDI RINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTV HSTDFEVKTDSTDHVKPKETENTKQPSKS Human TCR Delta2B: (SEQ ID NO: 35) AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIY REKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDTSSSS GTDKLIFGKGTRVTVEPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRI NLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHS TDFEVKTDSTDHVKPKETENTKQPSKS Human TCR Delta2C: (SEQ ID NO: 36) AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIY REKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDILGDK GNTDKLIFGKGTRVTVEPRSQPHTKPSVFVMKNGINVACLVKEFYPKDIR INLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVH STDFEVKTDSTDHVKPKETENTKQPSKS Cynomolgus Monkey TCR Delta2: (SEQ ID NO: 37) AVELVPEHQTVIVSVGDPATLKCSMKGEAISNYYINWYRKTQGNTMTFIY REKGIYGPGFKDNFQGDIDTEENQAVLKILAPSERDEGSYYCASDILSWV DSYTDKLIFGKGTRVTVEPKRQPHTKPSVFVMKNGTNVACLVKDFYGKDI RINLESSKKITEFDPAIVVSPSGKYNAVKLGQYADSNSVICSVQHNKEVV YSTDFEVKINSTDHLKPTETENTKQPSKS Human TCR Gamma3: (SEQ ID NO: 38) SSNLEGRIKSVTRQTGSSAEITCDLTVINTFYIHWYLHQEGKAPQRLLYY DVSTARDVLESGLSPGKYYTHTPRRWSWILRLQNLIENDSGVYYCATWDR PLNAWIKTFAKGTRLIVISPDKQLDADVSPKPTIFLPSIAETKLQKAGTY LCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEK SLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN Human TCR Gamma4: (SEQ ID NO: 39) SSNLEGRIKSVIRQTGSSAEITCDLAEGSTGYIHWYLHQEGKAPQRLLYY DSYTSSVVLESGISPGKYDTYGSTRKNLRMILRNLIENDSGVYYCATWDE KYYKKLFGSGTTLVVTDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLL EKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDK EHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN Human TCR Gamma5: (SEQ ID NO: 40) SSNLEGGIKSVIRPTRSSAEITCDLTVINAFYIHWYLHQEGKAPQRLLYY DVSNSKDVLESGLSPGKYYTHTPRRWSWILILRNLIENDSGVYYCATWDR LRKKLFGSGTTLVVTDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLE KFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKE HRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN Human TCR Gamma8: (SEQ ID NO: 41) SSNLEGRIKSVIRPTGSSAVITCDLPVENAVYTHWYLHQEGKAPQRLLYY DSYNSRVVLESGISREKYHTYASTGKSLKFILENLIERDSGVYYCATWDW GKKLFGSGTTLVVTDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEK FFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEH RCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN Human TCR Gamma9: (SEQ ID NO: 30) AGHLEQPQISSTKTLSKTARLECVVSGITISATSVYWYRERPGEVIQFLV SISYDGTVRKESGIPSGKFEVDRIPETSTSTLTIHNVEKQDIATYYCALW EAQQELGKKIKVFGPGTKLIITDKQLDADVSPKPTIFLPSIAETKLQKAG TYLCLLEKFFPDVIKIHWEEKKSNTILGSQEGNTMKTNDTYMKFSWLTVP EKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN Cynomolgus Monkey TCR Gamma: (SEQ ID NO: 42) AGHLEQPQISSTKMLSKTARLECVVSGVTISETSIYWYRERPGEVIQFLV CIFYDGTVKKESSIPSGKFEVDRIPKTSTSTLTIHNVEKQDIATYYCALW EVQQFGRKVKLFGPGTKLIITDKHLDADVSPKPTIFLPSIAETNLHKAGT YLCLLENFFPDVIKIHWQEKKSNTILGSQEGNTVKTNDTYMKFSWLTVPE KSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVTTMDPKDN Cynomolgus Monkey TCR Delta1A: (SEQ ID NO: 32) AQKVTQAQSSVSMPVEKAVTLNCQYETSSWSYDLFWYKQLPGKEMIFLIR QGSSEQNARDGRYSVNFKKEASFIALTISALQLEDSATYFCALRRPFTAQ LFFGKGTQLIVEPERQPHTKPSVFVMKNGTNVACLVKDFYPKDIRINLES SKKITEFDPAIVVSPSGKYNAVKLGQYADSNSVTCSVQHNKEVVYSTDFE VKTNSTDHLKPTETENTKQPSKS Cynomolgus Monkey TCR Delta1B: (SEQ ID NO: 33) AQKVTQAQSSVSMPVGKAVTLNCQYETSSWSYYLFWYKQLPGKEMIFLIH QGSSQQNARNGRYSVNFQKAASSITLTISALQLEDSATYFCALRERPPNP GPFVLGVYATAQLFFGKGTQLIVEPERQPHTKPSVFVMKNGTNVACLVKD FYPKDIRINLESSKKITEFDPAIVVSPSGKYNAVKLGQYADSNSVICSVQ HNKEVVYSTDFEVKINSTDHLKPTETENTKQPSKS Cynomolgus Monkey TCR Delta1C: (SEQ ID NO: 34) AQKVTQAQSSVSMPVEKAVTLNCQYETSWWSYDLFWYKQLPGKEMIFLIR QSSSEQNARDGRYSANFKKEASSKSFIALTISALQLEDSATYFCALPLQV RGPTGGIRVYDKLIFGKGTRVTVEPKRQPHTKPSVFVMKNGTNVACLVKD FYPKDIRINLESSKKITEFDPAIVVSPSGKYNAVKLGQYADSNSVICSVQ HNKEVVYSTDFEVKINSTDHLKPTETENTKQPSKS Synthetic Antibody Generation

Antibody clones having intended binding specificity were isolated from a phage-display library in the Fab format, following the design of an earlier-described library (Miller et al., PloS One. 2012, 7:e43746) with improvements. Phage library sorting was performed essentially following published procedures (Miller et al., PloS One. 2012, 7:e43746; Fellouse et al., J Mol Biol. 2007, 373:924-940). To enrich clones binding to the common regions of the delta1 chain, TCR samples consisting of different delta1 chains were used in successive rounds of library sorting. To eliminate clones binding to the delta2 chain or gamma chain, negative sorting using the sample consisting of the delta2 chain was also incorporated. To eliminate clones binding to the Fc portion of the TCR construct, the purified Fc was used as a competitor during sorting. Four rounds of library sorting were performed.

Binding to TCR clones was determined by phage ELISA (Sidhu et al., Methods Enzymol. 2000, 328:333-363.). Biotinylated TCR samples (“targets”; TCR (Delta1A/Gamma9), TCR (Delta1B/Gamma9), TCR (Delta1C/Gamma9), TCR (Delta2/Gamma9) or Fc) were immobilized to neutravidin-coated wells and blocked with an excess of biotin. The wells were incubated with phages displaying single Fab clones. Bound phages were detected with HRP-conjugated anti-M13 phage antibody.

From enriched pools of antibody clones, 10 clones bound to all three Delta1-containing TCRs but not to the Delta2-containing TCR (specificity exclusive to Delta1), one clone bound to both the Delta1- and Delta2-containing TCRs, and one showed no binding to any of the TCRs tested (FIG. 1). The amino acid sequences of their V_(H) and V_(L) chains (V_(H)/V_(L)) were deduced by determining the DNA sequences of the Fab genes in the phage clones and are reported herein as SEQ ID NO:1/SEQ ID NO:2, SEQ ID NO: 3/SEQ ID NO: 4, SEQ ID NO: 5/SEQ ID NO: 6, SEQ ID NO: 7/SEQ ID NO: 8, SEQ ID NO: 9/SEQ ID NO: 10, SEQ ID NO: 11/SEQ ID NO: 12, SEQ ID NO: 13/SEQ ID NO:14, SEQ ID NO: 15/SEQ ID NO: 12, SEQ ID NO: 17/SEQ ID NO: 18, SEQ ID NO: 19/SEQ ID NO: 12, SEQ ID NO: 21/SEQ ID NO: 12, and SEQ ID NO: 23/SEQ ID NO: 24.

IgG Protein Production

The genes for the antibody clones described above were transferred into mammalian expression vectors for human IgG production. The proteins were produced by transient transfection of ExpiCHO cells (ThermoFisher) and purified using Protein G Sepharose chromatography followed by Superdex 5200 or ResourceS chromatography (GE Healthcare).

Example 2: Characterization of Anti-Delta1 Antibody Clones

Epitope Binning

Whether the antibody clones bind to distinct (non-overlapping) epitopes in TCRs was examined using competition phage ELISA. Purified Delta1-6, -13 and -17 clones were used as competitors. Delta1-6 and Delta1-13 exhibited similar inhibition profiles against the 12 clones, whereas Delta1-17 had a distinct profile (FIG. 2). These results indicate that the identified antibodies bind to two or more distinct epitopes within gamma/delta1 TCR.

Affinity Measurements

The affinity of the antibodies was assessed using surface plasmon resonance (SPR). A biotinylated TCR sample was immobilized on an Avicap chip (Pall ForteBio) that had been preloaded with neutravidin (ThermoFisher). IgG samples were flowed using the OneStep method on a Pioneer SPR instrument (Pall ForteBio). The analyzed IgG samples had dissociation constant (K_(D)) values in the low nanomolar range to their respective targets (FIG. 3).

Inhibition of Gamma/Delta T Cell-Mediated Activation of T Cells

The effects of Delta1-17 IgG on the activity of gamma/delta T cells in inhibiting T cell activation were examined. Unstimulated conventional T cells from a blood sample of a colon cancer patient were stimulated and subsequently treated with gamma/delta T cells isolated from either blood or tumor sample of the patient and Delta1-17 IgG. Both types of gamma/delta T cells reduced the level of T cell activation; however, the addition of Delta1-17 IgG increased the level of T cell activation markers, indicating this antibody blocked the inhibitory function of gamma/delta T cells (FIG. 4).

Thermostability

Purified proteins were incubated with SYPRO-Orange at temperatures ranging from 25° C. to 99° C. in 0.5° C. increments. Fluorescence was measured at each increment. The average change in fluorescence versus temperature (i.e., the first derivative) was plotted for the 40° C. to 90° C. range, and is shown in FIG. 5. The curves are vertically offset for clarity.

Gamma Chain Binding

The ability to bind to a number of exemplary anti-Delta1 antibodies described herein were performed using heterodimers containing Delta1C (SEQ ID NO: 28) and different gamma chains (including gamma 3, 4, 5, 8, or 9), which were expressed as Fc-fusion proteins. The gamma/delta heterodimers were immobilized on the wells of a microtiter plate and probed with Delta1-6, -8, -10, -13, -15, and -17 clones or an isotype control. As shown in FIGS. 6A-H, the Delta1-8 and -17 clones bound all the different gamma/delta pairs equally well, whereas the other clones binding to the gamma 9 chain, and in the case of Delta1-15 additionally to the gamma 3 chain.

Example 3: Spheroid Preparation and Microfluidic Culture of Patient Tumor Samples

Fresh tumor specimens (human patients) are received in media (DMEM) on ice and minced in a 10-cm dish (on ice) using sterile forceps and scalpel. Minced tumor is resuspended in DMEM (4.5 mmol/L glucose, 100 mmol/L Na pyruvate, 1:100 penicillin-streptomycin; Corning CellGro)+10% FBS (Gemini Bio-Products), 100 U/mL collagenase type IV (Life Technologies), and 15 mmol/L HEPES (Life Technologies). Samples are pelleted and resuspended in 10 to 20 mL media. Red blood cells (RBC) are removed from visibly bloody samples using RBC lysis buffer (Boston Bio-Products). Samples are pelleted and then resuspended in fresh DMEM+10% FBS and strained over 100-μm filter and 40-μm filters to generate S1 (>100 μm), S2 (40-100 μm), and S3 (<40 μm) spheroid fractions, which are subsequently maintained in ultralow-attachment tissue culture plates. S2 fractions are used for ex vivo culture. An aliquot of the S2 fraction is pelleted and resuspended in type I rat tail collagen (Corning) at a concentration of 2.5 mg/mL following the addition of 10×PBS with phenol red with pH adjusted using NaOH. pH 7.0-7.5 is confirmed using PANPEHA Whatman paper (Sigma-Aldrich). The spheroid-collagen mixture is then injected into the center gel region of a 3-D microfluidic culture device as described in Jenkins et al., Cancer Discov. 2018 February; 8(2):196-215; Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids, the contents of which is herein incorporated by reference in its entirety. Collagen hydrogels containing patient-derived organotypic tumor spheroids (PDOTS) are hydrated with media with or without anti-γδ monoclonal antibodies after 30 minutes at 37° C.

In some cases, to test synergy with checkpoint inhibitors or other immunotherapy agents, PDOTS are treated with anti-PD-1 (pembrolizumab, 250 μg/mL), anti-CTLA4 (ipilimumab, 50 μg/mL), or combination (250 μg/mL pembrolizumab+50 μg/mL ipilimumab). For indicated PDOTS studies, anti-human PD-L1 is atezolizumab at 600 μg/mL+human IFNγ.

Immune profiling is performed by flow cytometry as described in Jenkins et al.

Example 4: Co-Culture Preparation, Treatment, and Analysis

In vitro culture assays using cells from a pancreatic cancer patient were performed to examine the effects of γδ T cells on T cells. Blood T cells were cultured alone or co-cultured with intratumoral or blood γδ T cells. Blood T cells were activated by ligation to CD3/CD28, resulting in an increase of TNF-α measured by FACS. As shown in FIG. 11A, co-culture of activated T cells with blood and tumor γδ T cells (bars 3 and 4, respectively) showed a significantly suppressed TNF-α signal, as compared to activated T cells (bar 2). Bar 1 represents inactivated T cells. The results demonstrate that γδ T cells from pancreatic cancer patients are immunosuppressive.

In an additional study, fresh colorectal tumor was excised from a patient at surgery and processed to produce 3-dimensional organoid cultures containing all tumor components, including immune cells. The patient's tumor representative organoid was treated with 100 nmol of an anti-delta1 mAb (using Delta1-17 as an example) for 72 hours, and then CD8⁺ T cells were activated. Percentage of TNF-α+ expressing (activated)CD8⁺ T cells were measured, and are shown in FIG. 11B. The control indicates baseline levels of CD8 T cell activity as measured by TNF-α expression. It was found that the anti-delta1 mAb blocked the immunosuppressive activity of γδ T cells within the organoid. Consequently, γδ T cell-mediated suppression of αβ T cells was relieved, resulting in activation of T cells.

Gamma delta T cell (GDT) and conventional T cells (αβ T cells) were sorted from human surgically resected tumor specimens and paired blood samples which were received fresh. For antibody-based T cell proliferation assays, blood or tumor CD3+ T cells were activated using a human T cell activation kit (130-091-441, Miltenyi Biotec, MA) in 96-well plates. In selected wells, Gamma delta T cells were added in a 1:5 GDT: T cell ratio. T cell activation, as indicated by TNF-α and IFNγ expression, was determined at 72 hr by flow cytometry. As shown in FIGS. 12A-12C, co-culture of activated αβ T cells with blood and tumor γδ T cells resulted in suppressed TNF-α and IFNγ signaling, demonstrating that the γδ T cells from three cancer patients (colorectal cancer, FIG. 12A, pancreatic cancer, FIG. 12B, and pancreatic cancer, FIG. 12C) are immunosuppressive.

Example 5: PDOTS Preparation, Treatment, and Analysis

PDOTS were prepared as described in Example 3 with slight modifications (Jenkins et al., 2017, Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids. Cancer Discov February; 8(2):196-215). Briefly, human surgically resected tumor specimens were received fresh in DMEM media on ice and minced in 10 cm dishes. Minced tumors were resuspended in DMEM+10% FBS with 100 U/mL collagenase type IV to obtain spheroids. Partially digested samples were pelleted and then re-suspended in fresh DMEM+10% FBS and strained over both 100 mm and 40 mm filters to generate S1 (>100 mm), S2 (40-100 mm), and S3 (<40 mm) spheroid fractions, which were subsequently maintained in ultra-low-attachment tissue culture plates. An aliquot of the S2 fraction was pelleted and resuspended in type I rat tail collagen at a concentration of 2.5 mg/mL following addition of 10×PBS with phenol red with pH adjusted using NaOH. An aliquot of the S2 fraction was pelleted and re-suspended in type I rat tail collagen and the spheroid-collagen mixture was then injected into the center gel region of the DAX-1 3D microfluidic cell culture chip (Aim Biotech, Singapore). After 30 min at 37° C., collagen hydrogels containing PDOTS were hydrated with media with indicated treatments including an anti-Delta1 antibody (using Delta1-17 as an example) and associated isotypes. PDOTS were kept in a sterile container and incubated in the standard cell culture incubator. PDOTS were collected after incubating for 3 days and further analyses of immune change were done by flow cytometry. As shown in FIGS. 13, 15-17, and 19, treatment of the PDOTS with the anti-Delta1 antibody described herein resulted in an increase in activated T cells in a colorectal cancer tumor sample (FIG. 13), a colorectal cancer liver metastasis tumor sample (FIG. 15), a second colorectal cancer liver metastasis tumor sample (FIG. 16), a hepatocellular carcinoma tumor sample (FIG. 17), and a second hepatocellular carcinoma tumor sample (FIG. 19). These results suggest that anti-Delta1 antibodies block the inhibitory function of the γδ T cells and allow activation of CD4+ and CD8+ T cells within the tumor spheroids.

Next, ability to activate T cells was compared between anti-Delta1 antibody and PD-1 antibody treatment. Treatment of PDOTS from a hepatocellular carcinoma tumor sample with the anti-Delta1 antibody described herein results in an increase in activated T cells as compared to the PD-1 antibody and the isotype control, suggesting that targeting gamma delta T cells is more effective than targeting a single check point molecule in blocking the inhibitory function of gamma delta T cells. (FIG. 18). Additionally, treatment of PDOTS from a second colorectal cancer tumor sample with an anti-PD1 antibody results in lower levels of T cells activation as compared to treatment with the or a combination of the anti-Delta1 antibody and the anti-PD1 antibody (FIG. 14).

Example 6: Cross Reactivity Assays

Delta1-17 is analyzed using the High-Spec® cross-reactivity assay on HuProt™ human proteome arrays, which contain the largest human protein collection on a single array. TCR (Delta2/Gamma9) is printed on the arrays as positive controls (at 1 μg/ml). Antibody samples are probed on HuProt arrays at 1 μg/ml. Delta1-17 recognized the expected target as the top hit, and showed high specificity.

Large-scale protein arrays are versatile and sensitive platforms for antibody specificity evaluation, screening of biomarkers, protein-protein, protein-small molecule, protein-DNA, protein-RNA, and protein-lipid interactions. These arrays can be used for the evaluation of protein-binding or affinity reagents, such as antibodies for in research and clinical applications, to assess specificity and find potential off-targets. The HuProt™ Human Proteome Microarray (CDI NextGen Proteomics) provides a large number of unique, full-length, individually purified human proteins on a single microscope slide, allowing interactions to be profiled in a high-throughput manner. The full-length recombinant proteins are expressed in the yeast S. cerevisiae, purified, and printed on glass slides in duplicate along with a set of control proteins (GST, BSA, histones, IgG, etc.).

Experimental Overview

The antibodies (Delta1-17 and isotype control (Ultra-LEAF-IgG1)) are probed at 1 μg/ml on the arrays and incubated at room temperature for 1 hour, and the secondary antibody is used as a negative control. After probing, the arrays are washed according to the protocol and probed with Alexa-647-anti-human IgG Fc gamma specific secondary antibodies under conditions optimized by CDI Labs for signal detection.

Data Analysis:

Non-specific hits that directly bind to the secondary antibodies are eliminated from the analysis of the samples. CDI software is used to quantify the specificity of each individual sample to specific proteins on the array based on Z Scores. The Z score is the average Z score of the duplicate spots of a given protein (each protein is printed in duplicate on a HuProt™ array). The Z score of each spot on a given array is calculated according to the algorithm Z=[F635−F635(avg)]/F635(std), wherein F635(avg) and F635(std) are the average and standard deviation of the F635 values of all spots on the array, respectively. The S score is the difference of the Z Scores of a given protein and the one ranked next to it. If the S score of the top hit is >3, the antibody is considered as highly specific against the top hit. F635 is the average foreground signal intensity of 2 replicate spots of a given protein in the detection channel (635 nm). B635 is the average background signal intensity of 2 replicate spots of a given protein in the detection channel (635 nm). Range includes 3 numbers, the F635 values of the 2 replicate spots and the difference between them. If the difference is too high (compared to the F635 value), it indicates the 2 spots are not consistent and the hit may be less reliable. The non-specific hits bound by the secondary antibody and the IgG1 control (Z score >3) are removed from the analysis of the samples.

Example 7: ADCC Activity of Delta1-17 IgG1

In antibody-dependent cell-mediated cytotoxicity (ADCC), effector cells lyse target cells on which antibodies have bound to specific antigens on the target cell membrane. The Fc effector function of Delta1-17 IgG1 was characterized and ADCC activity measured. A recombinant Jurkat T-cell line expressing firefly luciferase gene under the control of NFAT response elements with constitutive expression of human FcγRIIIa was used as an effector cell line.

γδ1 TCR sample was immobilized on an ELISA microplate and blocked. Delta1-17 IgG1 or isotype IgG1 was added to the well, incubated and then washed. Recombinant Jurkat T cells expressing firefly luciferase gene under the control of NFAT response elements with constitutive expression of human FcγRIIIa, high affinity (V158) variant were then added and incubated. Finally, a luciferase substrate was added to the well and measured the luciferace activity, which indicates the level of NFAT. Results are shown in FIG. 20, indicating that ADCC activity is dependent on the Delta1-17 antibody.

Example 8: ADCP Activity of Delta1-17 IgG1

Antibody-dependent cell-mediated phagocytosis (ADCP) is an important mechanism of action for antibodies that act partially or completely though phagocytosis. In this scenario, antibodies mediate uptake of specific antigens by antigen presenting cells. ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells, through FcγRIIa, FcγRI, and FcγRIIIa, of which FcγRIIa (CD32a) on macrophages represent the predominant pathway. In the ADCP assay being employed, THP-1 cells (human monocytic cell line derived from an acute monocytic leukemia patient) are used to measure ADCP.

Fluorescently labeled beads were conjugated with the target antigen, then incubated with the test antibody. THP-1 cells were then added to the plate to allow their binding to the Fc portion of antibodies bound to antigen-coated fluorescent beads. Antibody binding to the beads and engagement of the Fc-receptor results in an uptake of the beads by the THP-1 cells through a phagocytic mechanism. Phagocytosis events are analyzed using flow cytometry. Total amount of fluorescence in each cell-representing the number of beads phagocytosed—and the percentage of fluorescence-positive cells-representing the frequency of phagocytosis—are measured. Results are shown in FIG. 20, indicating that ADCP activity is dependent on the Delta1-17 antibody.

Other Embodiments

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. 

What is claimed is:
 1. A method of inhibiting the activation of immunosuppressive γδ T cells in a subject, the method comprising administering to a subject having or suspected of having a solid cancer a pharmaceutical composition comprising an antibody that binds a delta 1 chain of a T cell receptor, wherein the pharmaceutical composition is administered to the subject in an amount effective to enhance an anti-tumor immune response, and wherein the antibody comprises: a heavy chain comprising a heavy chain variable region (VH), wherein the VH comprises a heavy chain complementarity determining region 1 (CDR1) set forth as SEQ ID NO: 43, a heavy chain complementary determining region 2 (CDR2) set forth as SEQ ID NO: 57, and a heavy chain complementary determining region 3 (CDR3) set forth as SEQ ID NO: 77 and a light chain comprising a light chain variable region (VL), wherein the VL comprises a light chain complementarity determining region 1 (CDR1) set forth as SEQ ID NO: 46, a light chain complementary determining region 2 (CDR2) set forth as SEQ ID NO: 47, and a light chain complementary determining region 3 (CDR3) set forth as SEQ ID NO:
 78. 2. The method of claim 1, wherein the heavy chain comprises a heavy chain variable region (VH) set forth as SEQ ID NO: 23 and the light chain comprises a light chain variable region (VL) set forth as SEQ ID NO:
 24. 3. The method of claim 1, wherein the heavy chain further comprises a heavy chain constant region set forth as SEQ ID NO: 31, and/or wherein the light chain further comprises a light chain constant region set forth as SEQ ID NO:
 79. 4. The method of claim 2, wherein the heavy chain further comprises a heavy chain constant region set forth as SEQ ID NO: 31 and the light chain further comprises a light chain constant region set forth as SEQ ID NO:79.
 5. The method of claim 2, wherein the antibody comprises a heavy chain set forth as SEQ ID NO:84 and a light chain set forth as SEQ ID NO:83.
 6. The method of claim 1, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.
 7. The method of claim 1, wherein the antibody is a single chain antibody.
 8. The method of claim 1, wherein the antibody is a human antibody or a humanized antibody.
 9. The method of claim 1, wherein the antibody is an IgG molecule.
 10. The method of claim 9, wherein the IgG molecule is IgG1 or IgG4.
 11. The method of claim 2, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.
 12. The method of claim 2, wherein the antibody is a single chain antibody.
 13. The method of claim 2, wherein the antibody is a human antibody or a humanized antibody.
 14. The method of claim 1, wherein the subject is a human patient.
 15. The method of claim 1, wherein the subject is a human patient having a solid tumor selected from the group consisting of pancreatic ductal adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer, glioblastoma, upper and/or lower gastrointestinal malignancy, squamous cell head and neck cancer, genitourinary cancer, carcinoid tumors, endometrial cancer, renal cancer, bladder cancer, prostate cancer, ovarian cancer, neuroendocrine cancer, adrenocortical cancer, and sarcomas.
 16. The method of claim 15, further comprising administering to the subject an agent selected from the group consisting of an inhibitor of a checkpoint molecule, an activator of a co-stimulatory receptor, an inhibitor of an innate immune cell target, a chemotherapeutic agent, an anti-hypertension agent, and a combination of any agents thereof.
 17. A method of treating a solid tumor, the method comprising administering to a subject having or suspected of having a solid tumor an effective amount of a pharmaceutical composition comprising an antibody that binds a delta 1 chain of a T cell receptor to enhance anti-tumor immune responses, wherein the antibody comprises a heavy chain comprising a heavy chain variable region (VH), wherein the VH comprises a heavy chain complementarity determining region 1 (CDR1) set forth as SEQ ID NO: 43, a heavy chain complementary determining region 2 (CDR2) set forth as SEQ ID NO: 57, and a heavy chain complementary determining region 3 (CDR3) set forth as SEQ ID NO: 77 and a light chain comprising a light chain variable region (VL), wherein the VL comprises a light chain complementarity determining region 1 (CDR1) set forth as SEQ ID NO: 46, a light chain complementary determining region 2 (CDR2) set forth as SEQ ID NO: 47, and a light chain complementary determining region 3 (CDR3) set forth as SEQ ID NO:
 78. 18. The method of claim 17, wherein the antibody comprises a heavy chain variable region set forth as SEQ ID NO: 23 and a light chain variable region set forth as SEQ ID NO:
 24. 19. The method of claim 18, wherein the antibody comprises a heavy chain constant region set forth as SEQ ID NO: 31 and a light chain constant region set forth as SEQ ID NO:
 79. 